Method for inducing immune tolerance using non-proliferative polymer-modified allogeneic leukocytes

ABSTRACT

This invention relates to cellular-based therapies for increasing the level of regulatory T cells (Treg) and/or decreasing the level of pro-inflammatory T cells (Th17) to induce anergy or immune tolerance. To provide these therapeutic effects, a non-proliferative allogeneic leukocyte population is contacted with another leukocyte population. The non-proliferative allogeneic leukocyte population is modified to bear on its surface a low-immunogenic biocompatible polymer so as to prevent pro-inflammatory allo-recognition with the latter leukocyte population. Cellular-based preparations and processes for achieving cellular therapy are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/CA2013/050547 filed Jul. 12, 2013,which claims priority from CA patent application 2782942, U.S.provisional patent application 61/670,636 and U.S. provisional patentapplication 61/670,694 all filed on Jul. 12, 2012. The entire contentsof each of the above-referenced disclosures is specifically incorporatedby reference herein without disclaimer.

TECHNOLOGICAL FIELD

This invention relates to the use of allogeneic non-proliferativeleukocytes covalently modified with a biocompatible polymer to augmentthe level of regulatory T (Treg) cells and/or decrease the level ofpro-inflammatory T cells for inducing of a state of immune tolerance oranergy in the treated subjects. These modified leukocytes are useful forthe treatment of various conditions associated with excessive immuneresponses, auto-immunity and/or inflammation.

BACKGROUND

Acute and chronic rejection of donor tissues and organs remains asignificant clinical problem in transplantation medicine. Moreover,autoimmune diseases in which one's own immune system recognizes “self”tissues as foreign can also be rejected by similar mechanisms. Tominimize or prevent rejection, the administration of immunosuppressiveagents is typically required. Acute and chronic rejection are primarilyT lymphocyte-mediated events that require allogeneic recognition of theforeign tissue and the subsequent proliferation of allo-responsive Tcells. Indeed, because of the central role of the T cell in rejection,it is the primary target of current immunosuppressive drugs (e.g.,cyclosporine A, FK506). In general, these pharmacologic agents targeteither the T cell activation (e.g., cyclosporine A that inhibits IL-2responsiveness) or the proliferation (e.g., methotrexate) of theallo-responsive T cells. However all of today's clinically approvedanti-rejection drugs are beset by chronic toxicity; consequently,significant research is underway to identify alternative means ofpreventing acute and chronic rejection.

A biomaterials approach to the prevention of allo-recognition is thepolymer modification of donor cells (e.g., erythrocytes, lymphocytes,and pancreatic islets) (Scott et al., 1997; Murad et al., 1999A; Muradet al., 1999B; Bradley et al., 2001; Chen et al., 2001; Chen et al.,2003; McCoy et al., 2005; Chen et al., 2006; Bradley et al., 2007;Sutton et al., 2010; Le et al., 2010). The modification of the surfaceof cells is made by the direct grafting of low immunogenicity polymersto the cell membrane. Previous studies have demonstrated that thepolymer modification of erythrocytes and lymphocytes resulted in theloss of allo-recognition both in vitro and in vivo. Moreover, incontrast to pharmacologic agents, the grafted polymer exhibited bothextremely low toxicity and immunogenicity.

It would be highly desirable to be provided with a cellular-basedpreparation capable of inducing a state of anergy or immunotolerance byincreasing the ratio of the level of regulatory T cells (such as Treg)to pro-inflammatory T cells (such as Th1 and Th17). The cellularpreparation could induce anergy or tolerance either by increasing Treglevels, decrease pro-inflammatory T cell levels or both. Thispreparation could be useful for treating, preventing and/or alleviatingthe symptoms associated to an abnormal/excessive immune condition, suchas an auto-immune disease, a response to a vaccine or a tissue/celltransplantation.

BRIEF SUMMARY

One aim of the present invention is to provide cellular-basedpreparations capable of inducing a state of anergy or immunotolerance byincreasing the ratio of the level of regulatory T cells (such as Treg)to the level of pro-inflammatory T cells (such as Th1 and Th17). Thecellular-based preparations can induce anergy or tolerance either byincreasing Treg levels, decrease pro-inflammatory T cell levels or both.These preparations are useful for treating, preventing and/oralleviating the symptoms associated to an abnormal/excessive immunecondition, such as an auto-immune disease, a response to a vaccine or atissue/cell transplantation. The cellular-based preparations andtherapies presented herewith concern the use of at least two distinctleukocyte populations which are considered allogeneic with respect toone another, wherein at least one of the leukocyte population isnon-proliferative and modified to bear on its surface a low-immunogenicbiocompatible polymer so as to prevent pro-inflammatory allo-recognitionbetween the two leukocyte populations. The two leukocyte populations canbe contacted in vitro, ex vivo or in vivo to induce anergy or tolerance.

In accordance with the present invention, there is provided a method ofincreasing a ratio of the level of regulatory T (Treg) cells to thelevel of pro-inflammatory T cells in a subject in need thereof. Broadly,the method comprises administering: (i) a cellular preparationcomprising a first leukocyte having a cytoplasmic membrane associated toa low-immunogenic biocompatible polymer (wherein the first leukocyte isallogeneic to the subject as well as being non-proliferative); (ii) acultured cellular preparation comprising a leukocyte from the subjectwhich has been obtained by culturing the leukocyte from the subject withthe first leukocyte and/or (iii) a supernatant of a cell culture of asecond leukocyte having a cytoplasmic membrane associated to thelow-immunogenic biocompatible polymer and a third leukocyte (wherein thesecond leukocyte is allogeneic to the third leukocyte and at least oneof the second or the third leukocyte is non-proliferative). The methodis to provide an increase in the ratio of the level of Treg cells to thelevel of pro-inflammatory T cells in the treated subject. In anembodiment, the leukocyte is irradiated to prevent it fromproliferating. In an embodiment, the cytoplasmic membrane of the firstleukocyte and/or the second leukocyte has a membrane-associated proteincovalently bound to the low-immunogenic biocompatible polymer. In anembodiment, the leukocyte from the subject and/or the third leukocytehas a cytoplasmic membrane associated to a low-immunogenic biocompatiblepolymer. In such embodiment, it is also contemplated that thecytoplasmic membrane of the leukocyte from the subject and/or of thethird leukocyte has a membrane-associated protein covalently bound tothe low-immunogenic biocompatible polymer. In yet another embodiment,the leukocyte described herein is a T cell (such as, for example, aCD4-positive or a CD8-positive T cell). In another embodiment, in thecultured cellular preparation, the leukocyte from the subject isexpanded in vitro (or ex vivo) prior to administration to the subject.In yet another embodiment, in the cultured cellular preparation, thefirst leukocyte is removed prior to the administration to the subject.In an embodiment of the cell culture supernatant, the second leukocyteor the third leukocyte is from the subject. In yet another embodiment,the low-immunogenic biocompatible polymer is a polyethylene glycol (suchas for example mPEG) and/or 2-alkyloxazoline (POZ). In still anotherembodiment, the increased ratio between the level of Treg cells and thelevel of pro-inflammatory T cells is for treating, preventing and/oralleviating the symptoms associated to an auto-immune disease afflictingthe subject (such as, for example, type I diabetes, rheumatoidarthritis, multiple sclerosis, psoriasis, lupus, immunethrombocytopenia, experimental autoimmune encephalomyelitis, autoimmuneuveitis, inflammatory bowel disease, scleroderma and/or Crohn'sdisease). In still another embodiment, the increased ratio between thelevel of Treg cells and the level of pro-inflammatory T cells is forpreventing the onset of an excessive immune reaction in the subject(such as, for example, an excessive immune reaction in response to theadministration of a vaccine). In a further embodiment, the increasedratio between the level of Treg cells and the level of pro-inflammatoryT cells is for preventing or limiting the rejection of transplantedcells or tissue of the subject. In another embodiment, the transplantedcells and/or tissue are allogeneic or xenogeneic to the subject.

In accordance with the present invention, there is provided acellular-based preparation for increasing a ratio of regulatory T (Treg)cells to pro-inflammatory T cells in a subject. The cellular-basedpreparation comprises (i) a cellular preparation comprising a firstleukocyte having a cytoplasmic membrane associated to a low-immunogenicbiocompatible polymer (wherein the first leukocyte is allogeneic to thesubject and is considered non-proliferative); (ii) a cultured cellularpreparation comprising a leukocyte from the subject which has beenobtained by culturing it with the first leukocyte and/or (iii) asupernatant of a cell culture of a second leukocyte having a cytoplasmicmembrane associated to the low-immunogenic biocompatible polymer and athird leukocyte (wherein the second leukocyte is allogeneic to the thirdleukocyte and at least one of the second or the third leukocyte isnon-proliferative). The cellular-based preparation can be admixed withan appropriate excipient prior to administration to the subject.Embodiments with respect to the type of non-proliferative cells, thelow-immunogenic biocompatible polymer, the first leukocyte, theleukocyte from the subject, the second leukocyte, the third leukocyte aswell as the various uses of the preparations have been described aboveand do apply herein.

In accordance with the present invention, there is provided the use ofthe cellular-based preparations described herein for increasing a ratioof regulatory T (Treg) cells to pro-inflammatory T cells in a subject.There is also provided the use of the cellular-based preparationsdescribed herein for the preparation of a medicament for increasing aratio of regulatory T (Treg) cells to pro-inflammatory T cells in asubject. The cellular-based preparation comprises (i) a cellularpreparation comprising a first leukocyte having a cytoplasmic membraneassociated to a low-immunogenic biocompatible polymer (wherein the firstleukocyte is allogeneic to the subject and is modified so as to preventits proliferation); (ii) a cultured cellular preparation comprising aleukocyte from the subject which has been obtained by culturing it withthe first leukocyte and/or (iii) a supernatant of a cell culture of asecond leukocyte having a cytoplasmic membrane associated to thelow-immunogenic biocompatible polymer and a third leukocyte (wherein thesecond leukocyte is allogeneic to the third leukocyte and at least oneof the second or the third leukocyte is non-proliferative). Thecellular-based preparation can be admixed with an appropriate excipientprior to administration to the subject. Embodiments with respect to thetype of non-proliferative cells, the low-immunogenic biocompatiblepolymer, the first leukocyte, the leukocyte from the subject, the secondleukocyte, the third leukocyte as well as the various uses of thepreparations have been described above and do apply herein.

In accordance with the present invention, there is provided a processfor increasing and/or for providing the ability of a cellular-basedpreparation to increase a ratio of Regulatory T (Treg) cells topro-inflammatory T cells in a subject. Broadly the process comprises (i)associating a low-immunogenic biocompatible polymer to a cytoplasmicmembrane of a first leukocyte and refraining the first leukocyte fromproliferating to obtain a first modified leukocyte (wherein the firstleukocyte is allogeneic to the subject), (ii) culturing the firstmodified leukocyte with a leukocyte from the subject to obtain acultured cellular preparation and/or (iii) associating thelow-immunogenic biocompatible polymer to a cytoplasmic membrane of asecond leukocyte to obtain a second modified leukocyte, culturing thesecond modified leukocyte with a third leukocyte (wherein the secondleukocyte is allogeneic to the third leukocyte and at least one of thesecond or third leukocyte is non-proliferative), isolating the cellculture supernatant to obtain a cell culture supernatant; and (iv)formulating the first modified non-proliferative leukocyte, the culturedcellular preparation or the cell culture supernatant for administrationto the subject (such as, for example, intravenous administration). Theformulating step can also encompass formulating the first modifiednon-proliferative leukocyte, the cultured cellular preparation or thecell culture supernatant in a vaccine. Embodiments with respect to typeof non-proliferative cells, the low-immunogenic biocompatible polymer,the first leukocyte, the leukocyte from the subject, the secondleukocyte, the third leukocyte as well as the various uses of thepreparations have been described above and do apply herein.

Throughout this text, various terms are used according to their plaindefinition in the art. However, for purposes of clarity, some specificterms are defined below.

Allogeneic cell. A cell is considered “allogeneic” with respect toanother cell if both cells are derived from the same animal species butpresents sequence variation in at least one genetic locus. A cell isconsidered “allogeneic” with respect to a subject if the cell is derivedfrom the same animal species as the subject but presents sequencevariation in at least one genetic locus when compared to the subject'srespective genetic locus. Allogeneic cells induce an immune reaction(such as a rejection) when they are introduced into an immunocompetenthost. In an embodiment, a first cell is considered allogeneic withrespect to a second cell if the first cell is HLA-disparate (orHLA-mismatched) with the second cell.

Allo-recognition. As it is known in the art, the term “allo-recognition”(also spelled allorecognition) refers to an immune response to foreignantigens (also referred to as alloantigens) from members of the samespecies and is caused by the difference between products of highlypolymorphic genes. Among the most highly polymorphic genes are thoseencoding the MHC complex which are most highly expressed on leukocytesthough other polymorphic proteins may similarly result in immunerecognition. These polymorphic products are typically recognized by Tcells and other mononuclear leukocytes. In the context of the presentinvention, the term “pro-inflammatory allo-recognition” refers to animmune response associated with the expansion of pro-inflammatory Tcells and/or the differentiation of naïve T cells into pro-inflammatoryT cells. Pro-inflammatory allo-recognition in vivo mediates cell ortissue injury and/or death and loss of cell or tissue function. Still inthe context of the present invention, the term “pro-tolerogenicallo-recognition” refers to an immune response associated with theexpansion of Treg cells and/or the differentiation of naïve T cells intoTreg cells. A pro-tolerogenic allo-recognition is usually consideredweaker than a pro-inflammatory allo-recognition. Further, an in vivopro-tolerogenic allo-recognition does not lead to significant cell ortissue injury and/or death nor loss of cell or tissue function.

Anergy and Tolerance. In the present context, the term “anergy” refersto a non-specific state of immune unresponsiveness to an antigen towhich the host was previously sensitized to or unsensitized to. It canbe characterized by a decrease or even an absence of lymphokinesecretion by viable T cells when the T cell receptor is engaged by anantigen. In the present context, the term “tolerance” refers to anacquired specific failure of the immunological mechanism to respond to agiven antigen, induced by exposure to the antigen. Tolerance refers to aspecific non-reactivity of the immune system to a particular antigen,which is capable, under other conditions, of inducing an immuneresponse. However, in the present context, the terms “anergy” and“tolerance” are used interchangeably since the compositions and methodspresented herewith can be used to achieve both anergy and tolerance.

Autologous cell. A cell is considered “autologous” with respect toanother cell if both cells are derived from the same individual or fromgenetically identical twins. A cell is considered “autologous” to asubject, if the cell is derived from the subject or a geneticallyidentical twin. Autologous cells do not induce an immune reaction (suchas a rejection) when they are introduced into an immuno-competent host.

Immunogenic cell. A first cell is considered immunogenic with respect toa second cell when it is able to induce an immune response in the lattercell. In some embodiment, the immune response is in vitro (e.g. a mixedlymphocyte reaction) or can be observed in vivo (e.g. in a subjecthaving the second cell and having received the first cell). The secondcell can be located in an immunocompetent subject. Preferably, theimmune response is a cell-based immune response in which cellularmediator can be produced. In the context of this invention, theimmunogenic cells are immune cells, such as white blood cells orleukocytes.

Immunogenic cell culture conditions. A cell culture is considered to beconducted in immunogenic conditions when it allows the establishment ofa pro-inflammatory immune response between two distinct and unmodifiedleukocytes (and, in an embodiment, allo-recognition). Preferably, thepro-inflammatory immune response is a cell-based immune response inwhich cellular mediator can be produced. For example, the cell cultureconditions can be those of a mixed lymphocyte reaction (primary orsecondary). When a cell culture is conducted in immunogenic conditionsbut with leukocytes which have been modified to prevent or limitpro-inflammatory allo-recognition, no pro-inflammatory immune responseis observed. However, when a cell culture is conducted in immunogenicconditions but with leukocytes which have been modified to preventallo-recognition, a non-inflammatory immune response can be observed(for example a differentiation of naïve T cells to Treg cells and/orexpansion of Treg cells).

Leukocyte. As used herein, a leukocyte (also spelled leucocyte) isdefined as a blood cell lacking hemoglobin and having a nucleus.Leukocytes are produced and derived from hematopoietic stem cells.Leukocytes are also referred to as white blood cells. Leukocytes includegranulocytes (also known as polymorphonuclear leucocytes), e.g.neutrophils, basophils and eosoniphils. Leukocytes also includeagranulocytes (or mononuclear leucocytes), e.g. lymphocytes, monocytesand macrophages. Some of the lymphocytes, referred to as T cells (orT-cell), bear on their surface a T-cell receptor. T cell are broadlydivided into cells expressing CD4 on their surface (also referred to asCD4-positive cells) and cells expressing CD8 on their surface (alsoreferred to as CD8-positive cells). Some of the lymphocytes, referred toas B cells (or B-cells), bear on their surface a B-cell receptor.

Low-immunogenic biocompatible polymer. As used herein, a“low-immunogenic polymer” refers to a polymer which is not or isunlikely to elicit an immune response in an individual. Thislow-immunogenic polymer is also capable of masking an antigenicdeterminant of a cell and lowering or even preventing an immune responseto the antigenic determinant when the antigenic determinant isintroduced into a subject. A “biocompatible polymer” refers to a polymerwhich is non-toxic when introduced into a subject. Exemplarylow-immunogenic biocompatible polymers includes, but are not limited to,polyethylene glycol (for example methoxypoly(ethylene glycol)),hyperbranched polyglycerol (HPG) and 2-alkyloxazoline (POZ).

Non-proliferative leukocyte. As used herein, the term “non-proliferativeleukocyte” refers to a leukocyte which has been modified to no longerbeing capable of cellular proliferative (e.g. performing at least onecomplete division cycle). In some embodiments, this modification may betemporary and the non-proliferative properties of a leukocyte may belimited in time. For example, when a leukocyte is modified from acontact with a pharmacological agent capable of limiting itsproliferation, the removal of the pharmacological agent from the cellculture can allow the leukocyte to regain its proliferative properties.In other embodiments, the modification is permanent and the modifiedleukocyte cannot regain its proliferative properties. For example, whena leukocyte is irradiated, it is not possible for it to regain itsproliferative properties. In the context of the present application, theexpressions “non-proliferative leukocyte” or “leukocyte limited fromproliferating” are used interchangeably.

Peripheral blood mononuclear cells (PBMC). This term refers to the cellpopulation recuperated/derived from the peripheral blood of a subject(usually a mammal such as a human). PBMC usually contains T cells, Bcells and antigen presenting cells.

Pharmaceutically effective amount or therapeutically effective amount.These expressions refer to an amount (dose) of a cellular preparationeffective in mediating a therapeutic benefit to a patient (for exampleprevention, treatment and/or alleviation of symptoms of animmune-associated disorder in which the ratio of Tregs topro-inflammatory T cells is low when compared to a (sex- andaged-matched) healthy subject). It is also to be understood herein thata “pharmaceutically effective amount” may be interpreted as an amountgiving a desired therapeutic effect, either taken in one dose or in anydosage or route, taken alone or in combination with other therapeuticagents.

Prevention, treatment and alleviation of symptoms. These expressionsrefer to the ability of a method or cellular preparation to limit thedevelopment, progression and/or symptomology of a immune-associateddisorder associated to an abnormal/excessive immune response (in whichthe ratio of Tregs to pro-inflammatory T cells is low when compared to a(sex- and aged-matched) healthy subject). Broadly, the prevention,treatment and/or alleviation of symptoms encompasses increasing thelevels of Treg cells and/or decreasing the levels of pro-inflammatory Tcells. A method or cellular-based preparation is considered effective orsuccessful for treating and/or alleviating the symptoms associated withthe disorder when a reduction in the pro-inflammatory state (whencompared to an untreated and afflicted individual) in the treatedindividual (previously known to be afflicted with the disorder) isobserved. A method or cellular-based preparation is considered effectiveor successful for preventing the disorder when a reduction in thepro-inflammatory state (when compared to an untreated and afflictedindividual) in the treated individual is observed upon an immunologicalchallenge (such as, for example, an antigenic challenge).

Pro-inflammatory T cells. In the present context, pro-inflammatory Tcells are a population of T cells capable of mediating an inflammatoryreaction. Pro-inflammatory T cells generally include T helper 1 (Th1 orType 1) and T helper 17 (Th17) subsets of T cells. Th1 cells partnermainly with macrophage and can produce interferon-γ, tumor necrosisfactor-β, IL-2 and IL-10. Th1 cells promote the cellular immune responseby maximizing the killing efficacy of the macrophages and theproliferation of cytotoxic CD8+ T cells. Th1 cells can also promote theproduction of opsonizing antibodies. T helper 17 cells (Th17) are asubset of T helper cells capable of producing interleukin 17 (IL-17) andare thought to play a key role in autoimmune diseases and in microbialinfections. Th17 cells primarily produce two main members of the IL-17family, IL-17A and IL-17F, which are involved in the recruitment,activation and migration of neutrophils. Th17 cells also secrete IL-21and IL-22.

Regulatory T cells. Regulatory T cells are also referred to as Treg andwere formerly known as suppressor T cell. Regulatory T cells are acomponent of the immune system that suppress immune responses of othercells. Regulatory T cells usually express CD3, CD4, CD8, CD25, andFoxp3. Additional regulatory T cell populations include Tr1, Th3,CD8⁺CD28⁻, CD69⁺, and Qa-1 restricted T cells. Regulatory T cellsactively suppress activation of the immune system and preventpathological self-reactivity, i.e. autoimmune disease. The critical roleregulatory T cells play within the immune system is evidenced by thesevere autoimmune syndrome that results from a genetic deficiency inregulatory T cells. The immunosuppressive cytokines TGF-β andInterleukin 10 (IL-10) have also been implicated in regulatory T cellfunction. Similar to other T cells, a subset of regulatory T cells candevelop in the thymus and this subset is usually referred to as naturalTreg (or nTreg). Another type of regulatory T cell (induced Treg oriTreg) can develop in the periphery from naïve CD4⁺ T cells. The largemajority of Foxp3-expressing regulatory T cells are found within themajor histocompatibility complex (MHC) class II restrictedCD4-expressing (CD4⁺) helper T cell population and express high levelsof the interleukin-2 receptor alpha chain (CD25). In addition to theFoxp3-expressing CD4⁺CD25⁺, there also appears to be a minor populationof MHC class I restricted CD8⁺ Foxp3-expressing regulatory T cells.Unlike conventional T cells, regulatory T cells do not produce IL-2 andare therefore anergic at baseline. An alternative way of identifyingregulatory T cells is to determine the DNA methylation pattern of aportion of the foxp3 gene (TSDR, Treg-specific-demthylated region) whichis found demethylated in Tregs.

Splenocytes. This term refers to the cell population obtained from thespleen of a subject (usually a mammal such as a rodent). Splenocytesusually comprise T cell, B cell as well as antigen presenting cells.

Syngeneic cell. A cell is considered “syngeneic” with respect to asubject (or a cell derived therefrom) if it is sufficiently identical tothe subject so as to prevent an immune rejection upon transplantation.Syngeneic cells are derived from the same animal species.

Viable. In the present context, the term “viable” refers to the abilityof a cell to complete at least one cell cycle and, ultimatelyproliferate. A viable cell is thus capable of proliferating. Byopposition, the term “non-viable” or “non-proliferative” both refer to acell which is no longer capable of completing at least one cell cycle.By comparison, the term “cycle arrest” refers to a cell which has beentreated to halt its cell cycle progression (usually with apharmacological agent) but which is still capable of re-entering thecell cycle (usually when the pharmacological agent is removed).

Xenogeneic cell. A cell is considered “xenogeneic” with respect to asubject (or a cell derived from the subject) when it is derived from adifferent animal species than the subject. A xenogeneic cell is expectedto be rejected when transplanted in an immunocompetent host.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings, showing by way ofillustration, a preferred embodiment thereof.

FIG. 1 shows diagrammatically the conditioned media protocol. Aprimary)(1°) two-way mixed lymphocyte reaction (MLR) was initiated usingtwo HLA-disparate populations consisting of unmodified orpolymer-grafted (1 mM SVAmPEG; 5 kDa) PBMC. Within the mPEG-MLR, onlyone donor population was PEGylated. At 72 h, the conditioned media fromthe wells were collected. Secondary)(2°) mixed lymphocyte reactionsusing control and PEGylated PBMC from the same donors were initiated. Amitogen (PHA) stimulation control was added to assure that the mediacollected would support proliferation. 1° MLR Conditioned media:1=Resting unmodified PBMC; 2=Resting mPEG-PBMC; 3=Control MLR; and4=mPEG-MLR. 1° MLR/2° MLR Cell Types or Stimulation: A=Resting PBMC;B=Resting mPEG PBMC; C=MLR; D=mPEG MLR; P=PHA stimulation.

FIG. 2 shows 1° mixed lymphocyte reaction (MLR) results. Primary)(1°media cytokine levels at 72 h. IL-2 (A), IFN-γ (B) IL-17A (C), TNF-α (D)and IL-6 (E), levels are significantly reduced in the PEGylated two-wayMLR utilizing modified and unmodified PBMC populations from HLAdisparate individuals. The cytokine profile (ng/mL) was analyzed usingthe BD cytometric bead array. Values shown are the mean±SD of a minimumof four independent experiments. Percent non-viable cells within thecontrol and PEGylated (SVAmPEG; 5 kDa) resting PBMC was assessed bypropidium iodine exclusion (F).

FIG. 3 shows 2° mixed lymphocyte reaction (MLR) results. Shown is theproliferation index (percent PBMC proliferation) of the secondary MLR (□resting PBMC, ● control MLR, ◯ mPEG MLR, ▪ PHA stimulation) that wereconducted in the indicated (x-axis) conditioned media. As shown,relative to all other conditioned media, the media from the 1° platecontrol MLR demonstrated a significant (p<0.01) pro-proliferative effectin the 2° MLR. This effect was noted on even resting PBMC andPHA-stimulated cells. In contrast, the 1° conditioned media from themPEG-MLR demonstrated a significant anti-proliferative effect in the 2°MLR. As noted by the lines connecting paired experiments, PEGylation ofone donor population resulted in reduced proliferation in allconditioned media experiments. No significant differences were notedbetween fresh media in a parallel secondary plate and the resting PBMCconditioned media. Shown are the individual results of four independentexperiments and the mean (line). PEGylated cells were modified with 1 mMSVAmPEG (5 kDa).

FIG. 4 illustrates the effects of the various conditioned media on thelevels of Treg and Th17. PEGylation of human lymphocytes resulted in asignificant in vitro immunomodulatory effects as noted by changes in thepercentage of Treg (A) and Th17 (B) T cell populations (dark graycolumn→resting PBMC; light grey column→control MLR; white column→mPEGMLR; hatched column→PHA stimulation). Results are also provided forpercent PBMC proliferation (line) for 2° plates having received 1°conditioned media (defined in x-axis). As shown, the 1° media from themPEG-MLR favored production of Treg cells and a decreased population ofTh17 lymphocytes. In contrast, the 1° media from the control MLRenhanced Th17 cell production and greatly inhibited Treg levels. Therelative ratio of Th17:Treg was highly correlated with lymphocyteproliferation as denoted by the right y-axis and the embedded linegraph. The high levels of Tregs in both the resting mPEG-PBMC and inmPEG-MLR correlated with low levels of proliferation. In contrast, anincreased level of Th17 cells was associated with the 1° media from thecontrol MLR and PHA stimulation. PEGylated cells were modified with 1 mMSVAmPEG (5 kDa). Percent PBMC proliferation is provided in the righty-axis and by line on both panels.

FIG. 5 illustrates Treg levels in the spleen (A), in the brachial lymphnodes (B) or in the blood (C) in function of time (hours post injection)following administration of donor splenocytes or control (Δ naïve; ▴soluble mPEG; □ syngeneic cells; ▪ mPEG syngeneic cells; ● allogeneiccells; ◯ mPEG allogeneic cells). PEGylation of allogeneic donor murinesplenocytes resulted in a significant in vivo immunomodulatory effectgiving rise to significantly elevated Treg lymphocytes. As noted, in allthree tissues, a significant (p<0.001 at 120 h) increase in Treglymphocytes over that observed in naïve mice was noted in mice receivingmPEG-modified allogeneic donor cells. In stark contrast, a significantdecrease in Tregs (relative to naïve mice) is noted in mice transfusedwith unmodified allogeneic splenocytes. In comparing the absolutedifference between the control PEGylated splenocytes (dotted area or Δd)the differential impact of donor cell PEGylation can be fullyappreciated. Importantly, as noted at 120 h, transfusion of solublemPEG, syngeneic cells or mPEG-syngeneic cells had no significant effecton the Treg lymphocyte population. The range observed in naïve mice isdenoted by the grey bars. PEGylated murine splenocytes were modifiedwith 1 mM SVAmPEG (20 kDa).

FIG. 6 illustrates Th17 levels in the spleen (A), in the brachial lymphnodes (B) or in the blood (C) in function of time (hours post injection)following administration of donor splenocytes or control (Δ naïve; ▴soluble mPEG; □ syngeneic cells; ▪ mPEG syngeneic cells; ● allogeneiccells; ◯ mPEG allogeneic cells). PEGylation of allogeneic donor murinesplenocytes resulted in a significant in vivo immunomodulatory effect asevidenced by baseline levels of Th17 lymphocytes. As shown, unmodifiedallogeneic splenocytes resulted in a dramatic increase (p<0.001 at alltime points >24 h) in Th17 lymphocytes. However, PEGylation of theallogeneic donor cells completely abrogates this increase and the Th17levels stay in the range seen in naïve mice (grey area). In comparingthe absolute difference between the control and PEGylated splenocytes(dotted area or Δd) the differential impact of donor cell PEGylation canbe fully appreciated. Importantly, as noted at 120 h, transfusion ofsoluble mPEG, syngeneic cells or mPEG-syngeneic cells had no significanteffect on the Th17 lymphocyte population. The range observed in naïvemice is denoted by the grey bars. PEGylated murine splenocytes weremodified with 1 mM SVAmPEG (20 kDa).

FIG. 7 shows the ratio of Treg/Th17 levels five days followingadministration of donor splenocytes. PEGylation of allogeneic donormurine splenocytes resulted in a significant in vivo immunomodulatoryeffect. The panels in (A) show the ratio of Treg/Th17 levels in spleen(A1), “brachial” lymph node (A2), and peripheral blood (A3). The graphin (B) compares the ratio when non-modified allogeneic cells (rightside) or PEGylated allogeneic cells (left side) are administered. *denotes statistical significance (p<0.001).

FIG. 8 shows the long-term immunomodulatory effects of PEGylation ofdonor cells. The immunomodulatory effects of the PEGylated splenocytesis long lived and prevents changes in Treg and Th17 levels consequent torechallenge with unmodified allogeneic cells. Results are shown forpercentage of Tregs (upper panels) and Th17 cells (lower panels) in thespleen (right panels), brachial lymph nodes (middle panels) andperipheral blood (left panels) for mice transfused with allogeneicsplenocytes (▪) and mPEG allogeneic splenocytes (□). Thirty dayspost-transfusion with mPEG allogeneic splenocytes (◯), mice stilldemonstrated significantly elevated Treg levels demonstratingpersistence of the immunomodulation. When mice previously challengedwith mPEG-allogeneic splenocytes were rechallenged 30 days later withunmodified allogeneic splenocytes (●) no decrease in Treg or increase inTh17 cells were observed demonstrating tolerance/anergy. Shaded area onthe graph indicate Treg and Th17 levels in naïve mice. PEGylated murinesplenocytes were modified with 1 mM SVAmPEG (20 kDa).

FIG. 9 shows that immunomodulation is not haplotype-specific. Initialone-way MLR (◯) was conducted and consisted of C57Bl/6 (H-2b)splenocytes challenged with unmodified or PEGylated irradiated Balb/c(H2-d) splenocytes. Following 48 h of challenge, duplicate samples werechallenged with unmodified-non-irradiated C3H (H-2k) splenocytes(two-way MLR or ●). Results are shown as ³H-thymidine incorporation infunction of polymer (mPEG 5 kDa) grafting concentration (in mM). Theaddition of the fresh responder cells from a third, H2-disparate mousestrain (C3H), at 48 h did not reverse the attenuation of proliferationin responder cells co-incubated with irradiated, cmPEG-modified Babl/csplenocytes. In contrast, the proliferation in the control (0 mM) MLRwas significantly (p<0.001) enhanced by the addition of the C3Hsplenocytes (ΔC3H). The data shown represented the co-culturing of5.12×10⁶ C57Bl/6 splenocytes with 5.12×10⁶ irradiated, mPEG-derivitizedBalb/c splenocytes. After 48 h of incubation, fresh C3H responder cellswere added to duplicate wells. The results were expressed as the averagemean±standard deviation of triplicate samples from a representativeexperiment. PEGylated murine splenocytes were modified with theindicated concentrations (mM) of activated mPEG (5 kDa). For comparativepurposes, the anti-proliferative dose-response effect of cyclosporine A(CSA; which induces a pharmacologically-induced anergy) in a one-waymurine MLR under the same experimental condition is shown in the insert.

FIG. 10 provides an hypothetical representation of cellular-mediatedimmune modulation. (A) Current immunomodulation therapy almostexclusively targets the recipient's immune system and does not addressthe inherent antigenicity and immunogenicity of allogeneic tissues.Response to non-self is in large part mediated by cell-cell interactionsbetween Antigen Presenting Cells (APC; e.g., dendritic cells) and naïveT lymphocytes (Thp). This cell-cell interaction is characterized byadhesion, allorecognition and co-stimulation events. Consequent toallorecognition, cytokine/chemokine burst occurs followed byproliferation of pro-inflammatory T cells (e.g., CTL, Th17, Th1populations), immunoglobulin production and decreased evidence ofregulatory T cells (Treg). Current therapeutic agents are primarilycytotoxic agents preventing T cell activation (e.g., cyclosporine andrapamycin) or T cell proliferation (e.g., methotrexate, corticosteroids,azathiaprine). Additionally some blocking antibodies have beeninvestigated. (B) In contrast, polymer modification of donor PBMCresults in loss of appropriate cell-cell interaction leading to loss ofthe cytokine burst, decreased/absent proliferation, evidence ofapoptosis of alloresponsive T cells and increased levels of Regulatory T(Treg) cells that, in aggregate, provides a tolerogenic/anergic stateboth in vitro and in vivo. Shown with the schematic is a DNA ladderinggel of an unmodified MLR (A) and a PEGylated MLR (B) showing enhancedapoptosis consequent to PEGylation. Size of T cell population denotesincrease or decrease in number. Size of B cell indicates antibodyresponse.

FIG. 11 illustrates significant changes in the levels of Th17 and Treglymphocytes are noted in the spleen (upper panels), brachial lymph node(middle panels) and pancreatic lymph nodes (lower panels) uponconversion of NOD mice from non-diabetic (left panels) to diabetic(right panels). These changes are characterized by dramaticallyincreased Th17 (in the spleen, from 0.03 to 3.84%; in the brachial lymphnode from 0.01% to 0.67%; in the pancreatic lymph node from 0.05% to1.05%) and significantly decreased Treg (in the spleen, from 16.5% to2.0%; in the brachial lymph node from 11.8% to 1.8% and in thepancreatic lymph node, from 12.7% to 4.1%) lymphocytes. Tregs: *,p<0.001 from non-diabetic NOD mice. Th17: ** p<0.001 from non-diabeticNOD mice.

FIG. 12 illustrates cellular proliferation in a 2-way MLR of PEGylatedor POZylated cells at day 10. Results are shown for the mPEG-MLR (▪) andPOZ-MRL (□) as a percentage of proliferation (with respect to theproliferation of the control MLR; i.e., 0 mM) as a function of graftingdensity.

FIG. 13 illustrates the immunomodulatory effects of allogeneic andmPEG-allogeneic splenocytes upon injection in mice. Carrier (PBS),allogeneic splenocytes (SPL) or mPEG allogeneic splenocytes (mPEG-SPL)were injected in mice. (A) In vivo apoptosis is provided as percentageof apoptotic cells (e.g., Annexin V-positive cells) in in the spleen(grey bars) or the lymph node (white bars) in function of type ofinjection (PBS=control, SPL=unmodified allogeneic splenocytes,mPEG-SPL=mPEG allogeneic splenocytes). (B) Percentage of CD4-positivecells having a depolarized mitochondria in the spleen (grey bars) or thelymph node (white bars) in function of type of injection (PBS=control,SPL=unmodified allogeneic splenocytes, mPEG-SPL=mPEG allogeneicsplenocytes). (C) Percentage of intracellular IL-10-positive andCD4-positive cells in the spleen (grey bars) or the lymph node (whitebars) in function of type of injection (PBS=control, SPL=unmodifiedallogeneic splenocytes, mPEG-SPL=mPEG allogeneic splenocytes). (D) 5-dayweight gain (g) in mouse in function of type of injection (PBS=control,SPL=unmodifiedallogeneic splenocytes, mPEG-SPL=mPEG allogeneicsplenocytes). In (D), the SPL treated mice showed a loss of weightrelative to PBS of mPEG-SPL treated mice (0.64 g; approximately a 4%decrease in relative body weight). *=p<0.01 relative to PBS treatedanimal; #=p<0.01 relative to unmodified splenocytes.

FIG. 14 illustrates the effects of allogeneic splenocytes numbers andgrafting density on T cell differentiation in vivo. Percentage ofCD4-positive Tregs (white bars, percentage indicated on left y-axis) andTh17 cells (grey bars, percentage indicated on right y-axis) measured inresting Balb/c mice, mice having received unmodified allogeneic (e.g.C57BL/6) splenocytes (5, 20 or 40×10⁶ cells) or mice having receivedmPEG-modified (at a density of 0.5 mM, 1 mM or 4 mM) allogeneic (e.g.C57BL/6) splenocytes (5, 20 or 40×10⁶ cells). *=p<0.01 relative to naiveanimal; #=p<0.01 relative to animal administered unmodified splenocytes.

FIG. 15 illustrates the effects of allogeneic splenocytes on CD279expression of CD4-positive cells in vivo. Saline, syngeneic splenocytes(syngeneic), allogeneic splenocytes (allogeneic) or mPEG-allogeneicsplenocytes (mPEG-Allo) have been injected intravenously once (at day 0)or trice (at days 0, 2 and 5) in recipient mice. CD4-positive cells havebeen harvested 5 (◯) or 10 (●) days after the last injection. Thepercentage of CD4-positive and CD279-positive cells is shown in functionof type of injection (saline, syngeneic splenocytes, allogeneicsplenocytes or mPEG-allogeneic splenocytes) and number of injections(once=1, trice=3). (A) Results are shown for CD4-positive spleen cells.(B) Results are shown for CD4-positive lymph node cells. *=p<0.01relative to naïve (shaded area) animal; #=p<0.01 relative to animaladministered unmodified allogneic splenocytes.

FIG. 16 illustrates the effects of allogeneic splenocytes on thepercentage of Natural Killer (NK) cells in vivo. Saline, syngeneicsplenocytes (syngeneic), allogeneic splenocytes (allogeneic) ormPEG-allogeneic splenocytes (mPEG-Allo) have been injected intravenouslyonce (at day 0) or trice (at days 0, 2 and 5) in recipient mice. NKcells have been harvested 10 days after the last injection. Thepercentage of NK cells is shown in function of type of injection(saline, syngeneic splenocytes, allogeneic splenocytes ormPEG-allogeneic splenocytes), number of injections (once=1, trice=3) andlocation of the NK cells (●=spleen, ◯=brachial lymph node). Shaded arearefers to the percentage of NK levels in non-treated animals. *=p<0.01relative to naïve (shaded bar) animal; #=p<0.01 relative to animaladministered unmodified allogneic splenocytes.

FIG. 17 illustrates the effects of allogeneic splenocytes on the thymusin vivo. Saline, allogeneic splenocytes (Allo) or mPEG-allogeneicsplenocytes (mPEG-Allo) have been injected intravenously once inrecipient mice. Thymic cells have been harvested 5 days after theinjection. (A) The percentage of CFSE-positive donor cells (with respectto the total CD4-positive cells) is shown in function of type ofinjection (saline, allogeneic splenocytes or mPEG-allogeneicsplenocytes). White bar in mPEG-Allo sample represents the number ofdonor Tregs injected. (a) denotes CFSE positive donor cells showing thatno thymic microchimerisim is achieved in vivo (i.e., donor cells do notmigrate to, or survive in, the recipient thymus). (b) denotes theproliferative expansion of the donor Treg yielding thymicmicrochimerism. * p<0.01 relative to saline treated animal. # p<0.01relative to allogeneic treated animal. (B) The percentage of Treg cellsor CD25-positive cells (with respect to the total CD4-positive cells) isshown in function of type of injection (saline, allogeneic splenocytesor mPEG-allogeneic splenocytes). * p<0.01 relative to saline treatedanimal. # p<0.01 relative to allogeneic treated animal. (a) denotesdecrease in Treg in allogeneic treated animals. (b) denotes increase inTregs in mPEG-allogeneic treated animals over that of naïve animals. (c)denotes the proliferative expansion of the donor Treg yielding thymicmicrochimerism. * p<0.01 relative to saline treated animal. # p<0.01relative to allogeneic treated animal. (C) The percentage of Treg cells(white bars, percentage indicated in left y-axis, with respect to thetotal CD4-positive cells) and Th17 cells (grey bars, percentageindicated in right y-axis, in view of the total CD4-positive cells) isshown in function of type of injection (saline (naïve), allogeneicsplenocytes (Allo), gamma-irradiated allogneneic splenocytes (Ir-Allo),mPEG-allogeneic (mPEG-Allo) or gamma-irradiated allogeneic splenocytes(Ir mPEG-Allo)). Gamma-irradiated donor cells are incapable ofproliferation and are non-viable demonstrating that they can also beused to alter the immune response. Changes in T cell subsets in thymusare recipient-derived (e.g., CFSE-Negative, data not shown).

FIG. 18 illustrates that conditioned murine plasma modulates the Tregand Th17 differentiation levels in vivo. Conditioned murine plasma(obtained from donor mice 5 days post leukocyte transfer) wasadministered once or thrice to mice and Treg/Th17 levels were measuredin the spleen and the lymph nodes. (A) Results are shown as thepercentage of Treg cells (in function of CD4⁺ cells) (white bars, left yaxis) and as the percentage of Th17 cells (in function of CD4⁺ cells)(grey bars, right y axis) in the spleen of animals treated once (1) orthrice (3) with a control (Saline), a negative control conditionedplasma from animals having received saline (Plasma (Saline)), aconditioned plasma from animals having received unmodified allogeneicsplenocytes (Plasma (Allo)) or a condition plasma from animals havingreceived polymer-modified allogeneic splenocytes (Plasma (mPEG-Allo)).(B) Results are shown as the percentage of Treg cells (in function ofCD4⁺ cells) (white bars, left y axis) and as the percentage of Th17cells (in function of CD4⁺ cells) (grey bars, right y axis) in thebrachial lymph nodes of animals treated once (1) or thrice (3) with acontrol (Saline), a negative control conditioned plasma from animalshaving received saline (Plasma (Saline)), a conditioned plasma fromanimals having received unmodified allogeneic splenocytes (Plasma(Allo)) or a conditioned plasma from animals having receivedpolymer-modified allogeneic splenocytes (Plasma (mPEG-Allo)). *=p<0.01relative to saline control animal; #=p<0.01 relative to animaladministered with the unmodified allogeneic splenocytes (Plasma (Allo)).

FIG. 19 illustrates that conditioned murine plasma induces long-termchanges in cytokines expression levels in vivo. Conditioned murineplasma (obtained from donor mice 5 days post leukocyte transfer) wasadministered once or thrice to mice and intracellular cytokine positivecells were measured in the spleen and the lymph nodes. Results are shownas the percentage of intracellular cytokine positive cells (in functionof CD4⁺ cells) in the spleen of animals treated once (1) or thrice (3)with a negative conditioned plasma from animals having received saline(light grey bars), a conditioned plasma from animals having receivedunmodified syngeneic splenocytes (dark gray bars), a conditioned plasmafrom animals having received unmodified allogeneic splenocytes (hatchedbars) and a conditioned plasma from animals having receivedpolymer-modified allogeneic splenocytes (white bars). Results are shownfor IL-10, IL-2, TNF-α, IFN-γ and IL-4 either 30 or 60 days followingthe last administration of the conditioned serum or control. Similarresults have been obtained with the leukocytes obtained from thebrachial lymph nodes of these treated animals (data not shown).

FIG. 20 illustrates that conditioned murine plasma modulates multipleTreg subsets in vivo. Conditioned murine plasma (obtained from donormice 5 days post allogeneic leukocyte transfer) was administered miceand multiple Treg subset levels were measured in the spleen and thelymph nodes. Results are shown as the percentage of Treg subset (infunction of CD4⁺ cells) in the spleen and brachial lymph node of animalsadministered with a control (Saline), a negative control conditionedplasma from animals having received saline (Plasma (Saline)), aconditioned plasma from animals having received unmodified allogeneicsplenocytes (Plasma (Allo)) or a condition plasma from animals havingreceived polymer-modified allogeneic splenocytes (Plasma (mPEG-Allo)).Results are shown for Foxp3⁺ cells (white bars in the spleen, light graybars in the lymph node), CD25⁺ cells (hatches bars in the spleen, darkgrey bars in the lymph node) and CD69⁺ cells (horizontal hatched bars inthe spleen, diagonal hacthed bars in the lymph node).

FIG. 21 illustrates that conditioned murine plasma prepared from miceinjected with saline, allogeneic or mPEG allogeneic cells similarlymodulates Treg and Th17 differentiation levels in vivo. Conditionedmurine plasma (obtained from donor mice 5 days post leukocyte transfer)was administered to mice and Treg/Th17 levels were measured in thespleen, the lymph nodes and the blood five days after treatment. Resultsare shown for naïve animals (white bars) and animals receivingconditioned plasma prepared from animals having received saline (Plasma(Saline); light grey bars), animals having received unmodifiedallogeneic splenocytes (Plasma (Allo); dark grey bars) orpolymer-modified allogeneic splenocytes (Plasma (mPeg-All); hatchedbars). Results are shown as the percentage of Treg cells (in function ofCD4⁺ cells) in the spleen (A), the lymph node (B) or the blood (C).Results are also shown as the percentage of Th17 cells (in function ofCD4⁺ cells) in the spleen (D), the lymph node (E) or the blood (F).*=p<0.01 relative to saline control animal; #=p<0.01 relative to animaladministered with Plasma(Allo)-conditioned plasma.

DETAILED DESCRIPTION

In accordance with the present invention, there is providedcellular-based preparations for increasing the level of regulatory Tcells and/or decreasing the level of pro-inflammatory T cells forinducing a state of anergy, tolerance, loss of pro-inflammatory, and/orimmuno-quiescence in a subject in need thereof. The cellular-basedpreparations and therapies presented herewith concern the use of atleast two distinct leukocyte populations which are considered allogeneicwith respect to one another, wherein at least one of the leukocytepopulation is modified to bear on its surface a low-immunogenicbiocompatible polymer so as to prevent pro-inflammatory allo-recognition(but allowpro-tolerogenic allo-recognition) between the two leukocytepopulations. In the cellular-based preparations described herein, atleast one of the leukocyte population is considered non-proliferative.The cellular-based preparations can be obtained by modifying a firstleukocyte (which is considered immunogenic or allogeneic to the treatedsubject) to bear on its surface a low-immunogenic biocompatible polymerand to become non-proliferative. The cellular-based preparation can alsobe obtained by culturing the first leukocyte (bearing thelow-immunogenic biocompatible polymer) with a leukocyte from the subjectto obtain a cultured cellular preparation (wherein one of the twoleukocyte population is refrained from proliferating). Alternatively,the cellular-based preparation can be obtained by isolating the cellculture supernatant of a culture a second and a third leukocytes(wherein the second leukocyte is allogeneic to the third leukocyte andat least one of the second or third leukocyte is considerednon-proliferative) and the second leukocyte has been modified to bear onits surface a low-immunogenic biocompatible polymer.

As it will be shown below, polymer-based bioengineering of leukocyticcells provides a significant opportunity to modulate the responsiveness(i.e., immunoquiescent versus pro-inflammatory) of the recipient'simmune system. Without wishing to be bound to theory, it is hypothesizedthat polymer-modified white blood cells (e.g. leukocytes), andpreparations arising from such cells, can be used to induce Tregs and/orattenuate Th17/1 and NK (natural killer) cell upregulation, to preventthe pro-inflammatory immune response to allogeneic donor cells.Moreover, it is proposed that polymer-modified allogeneic white bloodcells (e.g. leukocytes) can be used therapeutically in various diseases(such as auto-immunity or an excessive immune response) to increase thelevels Treg cells and/or decrease proinflammatory effector cells, toultimately increase the ratio of regulatory T cells to pro-inflammatoryT cells thereby attenuating the incidence and/or severity of the diseasepathology.

The present invention provides experimental evidence that theadministration of polymer-modified (and in some embodiments, thePEGylated) of allogeneic human peripheral blood mononuclear cells (PBMC)or murine splenocytes can give rise to immunoquiescence (toleranceand/or anergy). This anergy was shown in vitro by conducting andanalyzing mixed lymphocyte reactions (MLR) and conditioned mediaexperiments for lymphocyte proliferation, differentiation and cytokineproduction. For lymphocyte differentiation, both in vitro and in vivo,the expansion/contraction of the regulatory T (Treg; favoring toleranceor anergy) and Th17 (pro-inflammatory and favoring allorejection)populations were quantitated. To more fully assess the systemic biologiceffect of polymer-mediated immune tolerance, in vivo murine studies werealso conducted to determine both the Treg and Th17 populationmodulations as well as whether differential effects were noted in thespleen, lymph nodes and blood of control and treated animals.

Methods for Modulating the Treg/Pro-Inflammatory T Cells Ratio

The present invention provides methods and cellular preparations forincreasing the ratio of the level of regulatory T cells with respect tothe level of pro-inflammatory T cells. In the present invention, theratio can be increased either by augmenting the level of regulatory Tcells in the subject or decreasing the level of pro-inflammatory T cellsin the subject. Alternatively, the ratio can be increased by augmentingthe level of regulatory T cells in the subject and decreasing the levelof pro-inflammatory T cells in the subject. When theTreg/pro-inflammatory T cells ratio is increased in a subject, it isconsidered that a state of anergy and/or of increased tolerance isinduced or present in the subject. The induction of a state of anergy orimmunotolerance in individuals experiencing an abnormally elevatedimmune reaction can be therapeutically beneficial for limiting thesymptoms or pathology associated with the abnormally elevated immunereaction. In some embodiments, it is not necessary to induce a state ofcomplete anergy or tolerance, a partial induction of anergy or tolerancecan be beneficial to prevent, treat and/or alleviate the symptoms of adisorder associated with a pro-inflammatory state (such as, for example,an auto-immune disease or an excessive immune response).

In order to increase the Treg/pro-inflammatory T cells ratio, anallogeneic cellular preparation can be administered to the subject in atherapeutically effective amount. In such instance, the cellularpreparation comprises a first leukocyte that has been modified with alow-immunogenic biocompatible polymer and that is considered to benon-proliferative. Prior to its modification with a low-immunogenicbiocompatible polymer, the first leukocyte is considered immunogenic(e.g. allogeneic) with respect to the subject because it is able toinduce an immune response (e.g. a cell-mediated immune response) whenadministered to the subject. As indicated above, it is possible todetermine if two cells are considered immunogenic with respect to oneanother by conducting conventional in vitro assays, such as a mixedlymphocyte reaction. It is also expected that MHC-disparate cells wouldbe considered immunogenic with respect to one another. In an embodiment,the first leukocyte can be xenogeneic to the subject. However, the firstleukocyte cannot be autologous or syngeneic to the subject. Prior to itsmodification, the first leukocyte is considered to be viable and capableof cellular proliferation. The first leukocyte can even be optionallyexpanded and or differentiated (e.g. from naïve to Treg) in vitro,however, in such embodiment, the first leukocyte is modified to becomenon-proliferative prior to its administration to the subject.

Alternatively, in order to increase the Treg/pro-inflammatory T cellsratio, a cultured cellular preparation can be administered to thesubject in a therapeutically effective amount. In order to do so, a thefirst leukocyte (modified with the low-immunogenic biocompatiblepolymer) is placed in contact in vitro with a leukocyte from the subjector a leukocyte syngeneic to the subject. One of the two leukocytepopulation (preferably the first leukocyte population) is modified,prior to the co-culture, to refrain from proliferating. The two cellpopulations are cultured under immunogenic conditions to provide acultured cellular preparation. However, since the first leukocytes hasbeen modified with a low-immunogenic biocompatible polymer, no or littlepro-inflammatory allo-recognition is observed in the cell culture. In anembodiment, one of the two population of leukocytes is not per se fromthe subject but is syngeneic to the subject. In an embodiment, theleukocytes from the subject (or syngeneic to the subject) are culturedunder conditions favoring the expansion (e.g. proliferation) and/ordifferentiation (e.g. naïve to Treg) prior to the administration to thesubject (either prior to, during or after the co-culture). In anotherembodiment, the first leukocytes are cultured under conditions favoringthe expansion (e.g. proliferation) and/or differentiation (e.g. naïve toTreg) prior to the co-culture. In some embodiments, it is preferable toremove the first leukocyte from the cultured cellular preparation priorto its administration to the subject. Methods of separating the twocellular populations are known to those skilled in the art and include,without limitation, cell sorting and magnetic beads. In an embodiment,the leukocyte from the subject can also be modified to comprise, on itscell surface, the low-immunogenic biocompatible polymer either prior tothe cell culture or after the cell culture (e.g. prior to theadministration to the subject).

An alternative way of increasing the Treg/pro-inflammatory T cell ratioconcerns the administration of the supernatant of a cell culture of asecond leukocyte (that has been modified with a low-immunogenicbiocompatible polymer prior to cell culture) and a third leukocyte(optionally modified with a low-immunogenic biocompatible polymer priorto or after cell culture). In such embodiment, at least one of thesecond or third leukocyte is limited from proliferating. In someembodiments, the cell culture supernatant can comprise leukocytes orleukocyte fractions (for example a part of the cytoplasmic membrane).The second leukocyte is considered immunogenic (e.g. allogeneic) withrespect to the third leukocyte because if the second leukocyte was notmodified and placed into contact with the third leukocyte, an immuneresponse (e.g. a cell-mediated immune response such as apro-inflammatory allo-recognition) would occur. It is possible todetermine if two cells are considered immunogenic with respect to oneanother by conducting conventional in vitro assays, such as the mixedlymphocyte reaction. It is also expected that MHC-disparate cells wouldbe considered immunogenic with respect to one another. In an embodiment,the second leukocyte cell can be xenogeneic to the third leukocyteHowever, the second leukocyte cannot be autologous or syngeneic to thethird leukocyte. In the methods and cellular compositions describedherein, it is possible that one of the second or third leukocyte besyngeneic or derived from the subject which will be treated. Inaddition, in other embodiments, both the second and/or third leukocytescan be considered allogeneic or xenogeneic to the subject which will betreated. The leukocytes are being cultured in conditions favoring invitro expansion and/or differentiation of naïve T cells toimmunomodulatory (e.g. Treg) cells of the leukocyte population. Prior tothe cell culture, one or both leukocyte populations can optionally beexpanded. Importantly, the cell culture supernatant, apart from beingoptionally filtered to remove cells and cellular debris, is notsubmitted to further extraction/size fractionation or specificenrichment of one of its components prior to its administration to thesubject. The cell culture supernatant thus comprises the conditionedmedia from the cell culture (e.g. cellular by-products such ascytokines).

An alternative way of increasing the Treg/pro-inflammatory Tcell ratioin a subject to be treated, it is also possible to administer theconditioned blood (preferably the plasma or the serum) of a test subject(usually an animal such as a mammal) that has been administered ortransfused with a first non-proliferative leukocyte modified to bear onits surface a low-immunogenic biocompatible polymer. Methods forrecuperating the blood or the blood fractions are known to those skilledin the art and usually include cell lysis and/or centrifugation. In someembodiments, this conditioned blood can comprise the first leukocyte ora derivative thereform (a part of the cytoplasmic membrane from thefirst leukocyte for example). The first leukocyte is consideredimmunogenic (e.g. allogeneic) with respect to the test subject becauseif the first leukocyte was not modified and transfused into the testsubject, an immune response (e.g. a cell-mediated immune response) wouldoccur. In another embodiment, the first leukocyte can be allogeneic orxenogeneic with respect to the test subject. However, the firstleukocyte cannot be autologous or syngeneic to the animal. In someembodiments, the first leukocyte can be allogeneic to the subject whichwill be treated. In alternative embodiment, the first leukocyte can besyngeneic or derived from the subject which will be treated with theconditioned blood.

In the context of the present invention, some of the leukocytes used inthe cellular preparations are both modified for bearing alow-immunogenic biocompatible polymer and being modified to no longer becapable of proliferation. The order in which the leukocytes are modified(modification with polymer and modification to prevent proliferation) isnot important. Leukocytes can be first modified to bear the polymer andthen modified to refrain from proliferating. Alternatively, theleukocytes can be first modified to refrain from proliferating and thenmodified to bear the polymer.

The leukocytes described herein can be derived from any animals, but arepreferably derived from mammals (such as, for example, humans and mice).

In the methods and cellular preparations provided herewith, the surfaceof the leukocyte is or can be modified with a low-immunogenicbiocompatible polymer. For some specific applications, it may bepreferable to modify the surface of the leukocyte with a single type oflow-immunogenic biocompatible polymer. However, for other applications,it is possible to modify the surface of the leukocyte with at least twodifferent types of low-immunogenic biocompatible polymers.

In order to achieve these modifications, the low-immunogenicbiocompatible polymer can be covalently bound to the cytoplasmicmembrane of the leukocyte and, in a further embodiment, amembrane-associated protein of the surface of the leukocyte or inserted,via a lipophilic tail, in the cytoplasmic membrane of the leukocyte.When the polymer is bound to a membrane-boud protein, themembrane-associated protein must have at least a portion which isaccessible on the external surface of the cytoplasmic membrane of theleukocyte for being covalently attached to the polymer. For example, themembrane-associated protein can be partially embedded in the cytoplasmicmembrane or can be associated with the external surface of the membranewithout being embedded in the cytoplasmic membrane. The low-immunogenicbiocompatible polymer can be covalently bound to a plurality ofmembrane-associated proteins. In an alternative or complementaryembodiment, the low-immunogenic biocompatible polymer can be inserted inthe cytoplasmic membrane by using a lipid-modified polymer.

In some embodiment, the low-immunogenic biocompatible polymer can bepolyethylene glycol (methoxy polyethylene glycol for example). Thepolyethylene glycol can be directly and covalently bound to amembrane-associated protein or, alternatively, a linker attaching thelow-immunogenic biocompatiable polymer can be used for attaching thepolymer to the protein. Exemplary linkers are provided in U.S. Pat. No.8,007,784 (incorporated herewith in its entirety). In alternativeembodiments, the low-immunogenic polymer can be POZ or HPG.

In the methods and cellular preparations provided herewith, theleukocytes can be mature leukocytes or be provided in the form of stemcells. For example, leukocytes can be obtained from isolating peripheralblood mononuclear cells (PBMC) from the subject. Optionally, the PBMCscan be differentiated in vitro into DC or DC-like cells. Alternatively,the leukocytes can be obtained from the spleen (e.g. splenocytes).Leukocytes usually include T cells, B cells and antigen presentingcells. In the methods and cellular preparations provided herewith, theleukocytes are not erythrocytes since the polymer-modified erythrocytesare not capable of increasing the ratio Treg/pro-inflammatory T cellswhen they were administered in a subject. However, traces oferythrocytes in the leukocytic preparations are tolerated (for example,less than about 10%, less than about 5% or less than about 1% of thetotal number of cells in the preparation).

Even though it is not necessary to further purify the leukocytes toconduct the method or obtain the cellular preparation, it is possible touse a pure cell population or a relatively homogenous population ofcells as leukocytes. This pure cell population and relative homogenouspopulation of cells can, for example, essentially consist essentially ofa single cell type of T cells, B cells, antigen presenting cells (APC)or stem cells. Alternatively, the population of cells can consistessentially of more than one cell type. The population of cells can beobtained through conventional methods (for example cell sorting ormagnetic beads). In an embodiment, when the population of cells consistof a single cell type (for example, T cells), the percentage of the celltype with respect to the total population of cells is at least 90%, atleast 95% or at least 99%. The relatively homogenous population of cellsare expected to contain some contaminating cells, for example less than10%, less than 5% or less than 1% of the total population of cells.

The cell culture supernatant used in the method or in the cellularpreparation can be obtained by co-culturing a second leukocyte cellularpopulation with a third leukocyte cellular population. It is alsopossible to co-culture a second leukocyte homogenous cell population(such as, for example, a T pure cell population or a substantially pureT cell population) with a third leukocyte preparation. It is alsocontemplated to culture a second leukocyte population with a thirdleukocyte population (such as, for example, a pure T cell population ora substantially pure T cell population).

In addition, and as indicated above, when the subject's own cells areused in the cell culture (to provide the culture supernatant), they canbe modified to be covalently bound to the low-immunogenic biocompatiblepolymer and cultured with the first leukocyte. Alternatively, they canremain unmodified (e.g. not covalently bound to the low-immunogenicbiocompatible polymer) and cultured with the first leukocyte which hasbeen modified to be covalently bound to the low-immunogenicbiocompatible polymer.

In the methods and preparations presented herewith, it is required toinhibit/limit the proliferation of at least one of the leukocytepopulation being contacted. For example, a leukocyte can betreated/modified prior to cell culture or its administration into thesubject in order to inhibit/limit the cell from proliferating in thesubject. For example, the cell can be irradiated (e.g. γ-irradiation)prior to its introduction in the subject or its introduction into aculture system. Upon irradiation, the leukocyte is not considered viable(e.g. capable of proliferation). In an embodiment, polymer grafting canaffect the leukocyte viability and can be used to refrain the leukocytefrom proliferating. Alternatively, leukocyte can be treated with apharmacological agent which halts cell cycle progression. Upon theadministration of such pharmacological agent, the leukocyte isconsidered viable since it can resume cellular proliferation when theagent is removed from the cell-containing medium. When the cell culturesupernatant is used in the method, it is important that only one of thetwo cell populations be inhibited/limited from proliferating and thatthe other cell population be able to proliferate.

The conditioned blood that can be used in the method can be obtained byadministering (preferably transfusing or intravenously administering),to a test subject, a first leukocyte considered allogeneic to the testsubject which has been modified with a low-immunogenic and biocompatiblepolymer. It is also possible to transfuse a first leukocytic homogenouscell population (such as, for example, a T pure cell population or asubstantially pure T cell population) to the subject. The blood isrecuperated from the subject after a time sufficient to induce in thesubject a state of anergy or tolerance. As indicated above, the firstleukocytic cellular preparation is inhibited/limited from proliferatingprior to administration to the animal.

As shown herein, the administration of the cellular preparations inducesa state of anergy or immune tolerance in the treated subject. In someembodiments, the state of anergy can persist long after theadministration of the cellular preparation or the cell culturesupernatant (as shown below, at least 270 days in mice). In an optionalembodiment, the state of anergy does not revert back to apro-inflammatory state upon a challenge with, for example, an immunogen(such as an immunogenic or allogeneic cell). Consequently, the methodsand cellular preparations described herein are useful for the treatment,prevention and/or alleviation of symptoms associated withabnormal/excessive immune responses and conditions associated thereto.

Autoimmunity arises consequent to an animal/individual's immune systemrecognizing their own tissues as “non-self”. Autoimmunity is largely acell-mediated disease with T lymphocytes playing a central role in“self” recognition and are, in many cases, also the effector cells. TheNon-Obese Diabetic (NOD) mouse is an inbred strain that exhibits thespontaneous development of a variety of autoimmune diseases includinginsulin dependent diabetes. The murine autoimmune diabetes developsaround 16 to 20 weeks of age and has been extensively used to study themechanisms underlying autoimmune-mediated diabetes, therapeuticinterventions and the effect of viral enhancers on disease pathogenesis.Diabetes develops in NOD mice as a result of insulitis, a leukocyticinfiltrate of the pancreatic islets. This can be exacerbated if mice areexposed to killed mycobacterium or other agents (Coxsackie virus forexample). Multiple studies have established that the pathogenesis ofdiabetes in the NOD mouse is very similar to that observed in human typeI diabetes (T1D) in that it is characterized by the breakdown ofmultiple tolerance pathways and development of severe insulitis of theislets prior to β-cell destruction. Moreover, T cells (including Th1,Th17 and Tregs) have been identified as key mediators of the autoimmunedisease process though other cells (NK cells, B-cells, DC andmacrophages) are also observed. Indeed, the NOD mouse model hastranslated into successful clinical human trials utilizing T-celltargeting therapies for treatment of many autoimmune diseases, includingT1D.

The loss of function arising from pro-inflammatory allo-recognition isexemplified by the destruction of the islets of Langerhans (insulinsecreting 11 cells) in the pancreas of the NOD mice leading to the onsetof Type 1 diabetes. In this context context, pro-tolerogenicallo-recognition is characterized by the protection and survival of theislets of Langerhans and the inhibition of diabetes in the NOD mouse.

Treatment of most autoimmune diseases is problematic and normallyfocused on addressing disease symptoms, not causation. Typically,treatment for chronic autoimmune disease is via systemic steroid (e.g.,dexamethasone) administration to induce a general immunosuppression andto act as an anti-inflammatory agent. It is believed that one mechanismof this immunosuppression may be the induction of Treg cells. Inaddition to steroids, the administration of IVIg (pooled, polyvalent,IgG purified from the plasma of >1 000 blood donors) can alsoeffectively treat some autoimmune diseases including immunethrombocytopenia (ITP). Interestingly, the onset of diabetes in NOD micecan also be delayed, but not fully blocked by administration of IVIg andthis may correlate with induction of Tregs (and/or IL-10). Hence, novelapproaches to increase Treg cells (and/or IL-10) while decreasinginflammatory T cell responses (e.g., Th17, NK cells) could be beneficialin treating autoimmune diabetes.

A state of anergy or immune tolerance can be considered therapeuticallybeneficial in subjects experiencing (or at risk of experiencing) anabnormal immune response, such as for example an auto-immune disease.Some auto-immune diseases are associated with either low levels of Tregsand/or elevated levels of pro-inflammatory T cells (such as Th17 and/orTh1). Such auto-immune diseases include, but are not limited to, type Idiabetes, rheumatoid arthritis, multiple sclerosis, lupus, immunethrombocytopenia, experimental autoimmune encephalomyelitis, auto-immuneuveitis, psoriasis inflammatory bowel disease, scleroderma and Crohn'sdisease. Because it is shown herein that the cellular preparations arebeneficial for increasing the ratio Tregs/pro-inflammatory T cells, itis expected that administration of the cellular preparations toafflicted subject will alleviate symptoms associated with theauto-immune disease.

A state of anergy or tolerance can also be considered therapeuticallybeneficial in subjects at risk of developing an abnormallyelevated/excessive immune response. Such abnormally elevated immuneresponse can be observed in subjects receiving a vaccine. For example,it has been shown that subjects receiving a respiratory syncytial virus(RSV) vaccine develop an excessive immune response. Such abnormallyelevated immune response can also be observed in subjects receiving atransplant (cells or tissues). In these conditions, the methods andcellular preparations can be applied to prevent or limit theelevated/excessive immune response. The cellular preparation, culturedcellular preparation or the cell culture supernatant can beco-administered with the vaccine or the transplant. Alternatively, thecellular preparation, the cultured cellular preparation or the cellculture supernatant can be administered prior to the administration ofthe vaccine or the introduction of the transplant to induce a state ofanergy or tolerance in the subject. In some embodiments, the first,second and/or third leukocyte can be syngeneic to the tissue/cell donor.

In the methods and cellular preparations described herein, it iscontemplated that the cellular-based preparations be optionallyadministered with other therapeutic agents known to be useful for thetreatment, prevention and/or alleviation of symptoms of conditionsassociated to an excessive/abnormal immune response, such as, forexample, cortisone, IL-10, IL-11 and/or IL-12.

Processes for Obtaining Cellular Preparations

The cellular preparations presented described herein can be obtained bycontacting two distinct and allogeneic leukocyte populations. One of thetwo leukocyte population is modified to bear on its surface (and, insome embodiment, to be covalently bound to) a low-immunogenicbiocompatible polymer. One of the two leukocyte population is modifiedto be refrained from proliferating. The two leukocyte populations arecontacted under conditions so as to limit (and in some embodimentsprevent) pro-inflammatory allo-recognition and to allow pro-tolerogenicallo-recognition.

It is important that the polymer used exhibits both low-immunogenicityand biocompatibility once introduced into a cell culture system oradministered to the test subject. It is shown below that polyethyleneglycol (particularly methoxypoly(ethylene glycol)) and POZ have beensuccessfully used to modify leukocytes and provide correspondingcellular preparations having the ability to increase a ratio of Tregcells to pro-inflammatory T cells in vitro and in vivo. Theseexperimental results suggest that other low-immunogenic biocompatiblepolymers can also be used to modify leukocytes. These otherlow-immunogenic biocompatible polymers include, but are not limited toan hyperbranched polyglycerol (HPG). In some embodiments, it ispreferable to use a single type of polymer to modify the surface ofleukocytes. In other embodiments, it is possible to use at least twodistinct types of polymers to modify the surface of the leukocyte.

In an embodiment, the low-immunogenic biocompatible polymer can becovalently associated with the membrane-associated protein(s) of theleukocyte by creating a reactive site on the polymer (for example bydeprotecting a chemical group) and contacting the polymer with theleukocyte. For example, for covalently binding a methoxypoly(ethyleneglycol) to the surface of a leukocyte, it is possible to incubate amethoxypoly(-ethylene glycol) succinimidyl valerate (reactive polymer)in the presence of the leukocyte. The contact between the reactivepolymer and the leukocyte is performed under conditions sufficient forproviding a grafting density which will prevent pro-inflammatoryallo-recognition and allow pro-tolerogenic allo-recognition. In anembodiment, the polymer is grafted to a viable leukocyte and underconditions which will retain the viability of the leukocyte. A linker,positioned between the surface of the leukocyte and the polymer, canoptionally be used. Examples of such polymers and linkers are describedin U.S. Pat. Nos. 5,908,624; 8,007,784 and 8,067,151. In anotherembodiment, the low-immunogenic biocompatible polymer can be integratedwithin the lipid bilayer of the cytoplasmic membrane of the leukocyte byusing a lipid-modified polymer.

As indicated above, it is important that the low-immunogenicbiocompatible polymer be grafted at a density sufficient for preventingpro-inflammatory allo-recognition and allow pro-tolerogenicallo-recognition). In an embodiment, the polymer is polyethylene glycol(e.g. linear) and has an average molecular weight between 2 and 40 KDaas well as any combinations thereof. In a further embodiment, theaverage molecular weight of the PEG to be grafted is at least 2, 3, 4,5, 10, 15, 20, 25, 30, 35 or 40 kDa. In another embodiment, the averagemolecular weight of the PEG to be granted is no more than 40, 35, 30,25, 20, 15, 10, 5, 4, 3, or 2 kDa. In another embodiment, the graftingconcentration of the polymer (per 20×10⁶ cells) is at least 1, 1.5, 2,2.5, 5, 6, 7, 8, 9 or 10 mM. In still another embodiment, the graftingconcentration of the polymer (per 20×10⁶ cells) is no more than 10, 9,8, 7, 6, 5, 2.5, 2, 1.5 or 1 mM. In order to determine ifpro-inflammatory allo-recognition occurs (or is prevented), varioustechniques are known to those skilled in the art and include, but arenot limited to, a standard mixed lymphocyte reaction (MLR), highmolecular weight mitogen stimulation (e.g. PHA stimulation) as well asflow cytometry (Chen and Scott, 2006). In order to determine if a weakpro-tolerogenic allo-recognition occurs (or is prevented), varioustechniques are known to those skilled in the art and include, but arenot limited to, the assessment of the level of expansion anddifferentiation of Treg cells and or prevention of Th17expansion/differentiation. In an embodiment, the polymer is selected andgrafted to the modified leukocyte to provide to the modified leukocyte apro-inflammatory/pro-tolerogenic allo-recognition substantially similarto the one observed in a mixed lymphocyte reaction between a firstleukocyte modified to be grafted with 20 kDa mPEG at a density of atleast 0.5 mM (and preferably 1 mM) per 20×10⁶ cells and incubated with asecond (unmodified) allogeneic leukocyte.

Before or after being modified with a low-immunogenic biocompatiblepolymer, the leukocyte can be modified to refrain them from beingproliferative. For example, the cell can be irradiated (e.g.γ-irradiation) prior to its introduction in the subject or itsintroduction into a culture system. Upon irradiation, the leukocyte isnot considered viable (e.g. capable of proliferation). In an embodiment,polymer grafting can affect the leukocyte viability and can be used torefrain the leukocyte from proliferating. Alternatively, leukocyte canbe treated with a pharmacological agent which halts cell cycleprogression. Upon the administration of such pharmacological agent, theleukocyte is considered viable since it can resume cellularproliferation when the agent is removed from the cell-containing medium.

As indicated above, the leukocytes can be inhibited/limited fromproliferating prior to their introduction in a cell culture oradministration to the test subject. For example, the first and/or secondleukocyte can be irradiated to halt its proliferation prior to itsadministration to the subject, its contact with the leukocyte from thesubject or its contact with the third leukocyte. In some embodiments,the first and/or second leukocyte can first be modified with thelow-immunogenic biocompatible polymer and then inhibited/limited fromproliferating. In other embodiments, the first and/or second leukocytecan first be inhibited/limited from proliferating and then modified withthe low-immunogenic biocompatible polymer.

To provide the cellular preparations described herewith, the leukocytesused can be mature leukocytes or be provided in the form of stem cells.For example, leukocytes can be obtained from isolating peripheral bloodmononuclear cells (PBMC) from the subject. Optionally, the PBMCs can bedifferentiated in vitro into dendritic (DC) or DC-like cells.Alternatively, the leukocytes can be obtained from the spleen (e.g.splenocytes). Leukocytes usually include T cells, B cells and antigenpresenting cells. For providing the cellular preparations, theleukocytes are not erythrocytes since the polymer-modified erythrocytesare not capable of causing a pro-tolerogenic allo-recognition whenadministered in a test subject. However, traces of erythrocytes in theleukocyte population used are tolerated (for example, less than about10%, less than about 5% or less than about 1% of the total number ofcells in the preparation).

Even though it is not necessary to further purify the leukocytes toprovide the cellular preparations, it is possible to use a pure cellpopulation or a relatively homogenous population of cells as leukocytes.This “pure” cell population and “relative homogenous population” ofcells can, for example, essentially consist essentially of a single celltype of T cells, B cells, antigen presenting cells (APC) or stem cells.Alternatively, the population of cells can consist essentially of morethan one cell type. The population of cells can be obtained throughconventional methods (for example cell sorting or magnetic beads). In anembodiment, when the population of cells consist of a single cell type(for example, T cells), the percentage of the cell type with respect tothe total population of cells is at least 90%, at least 95% or at least99%. The relatively homogenous population of cells are expected tocontain some contaminating cells, for example less than 10%, less than5% or less than 1% of the total population of cells.

The leukocytes can be obtained from any animals, but are preferablyderived from mammals (such as, for example, humans and mice). In anembodiment, the leukocytes can be obtained from a subject intended to betreated with the cellular preparations.

In an optional embodiment, to obtain the cellular preparation, the firstleukocyte (modified to bear the low-immunogenic biocompatible polymer)can be placed in cell culture (in immunogenic conditions) with aleukocyte from the subject (or a leukocyte syngeneic to the subject) andthe resulting cultured cellular preparation can be administered to thesubject in need thereof. The cultured cellular preparation comprises atleast the cultured leukocyte from the subject (or the cultured leukocytesyngeneic to the subject). In some embodiment, the cultured leukocytefrom the subject (or the leukocyte syngeneic to the subject) can bemodified (either prior to or after the cell culture) to bear thelow-immunogenic biocompatible polymer. In the co-culture, at least oneof the two leukocyte preparation is also modified to refrain fromproliferating. In additional embodiments, the cultured leukocytes can beexpanded and/or differentiated (from naïve to Treg) in vitro prior totheir administration to the subject (prior to, during or after theco-culture). The cultured cellular preparation can be formulated toinclude or exclude the first leukocyte.

When a co-culture system is used, it is possible to culture a firstleukocytic population (such as, for example a PBMC or splenocyte) with aleukocytic population from a subject (such as, for example a PBMC orsplenocyte). It is also possible to culture a first leukocyticrelatively homogenous cell population (such as, for example, a T cellpopulation) with a leukocytic population from a subject (such as, forexample a PBMC or splenocyte). It is also contemplated to culture afirst leukocytic population (such as, for example a PBMC or splenocyte)with a leukocytic relatively homogenous population of cells from thesubject (such as, for example, a T cell population). It is furthercontemplated to culture a first leukocytic relatively homogenous cellpopulation (such as, for example, a T cell population) with a leukocyticrelatively homogenous population of cells from the subject (such as, forexample, a T cell population). In some embodiments, the first leukocyteis cultured in a vessel which does allow physical contact with theleukocyte from the subject.

Usually, the cultured cellular preparation is obtained at least 24 hoursafter the initial contact between the first leukocyte and the leukocytefrom the subject. In some embodiment, the cultured cellular preparationis obtained at least 48 hours or at least 72 hours after the initialcontact between the first leukocyte and the leukocyte from the subject.In an embodiment, the cultured cellular preparation can be obtainedafter at least 24 hours of incubating a first leukocyte (for examplegrafted with a 20 kDa PEG at a density of at 1.0 mM) with the leukocytefrom the subject. When the incubation takes place in a 24-well plate,the concentration of each leukocyte population can be at least 1×10⁶cells.

In yet a further optional embodiment, to obtain the cellularpreparation, a polymer-modified second leukocyte can be placed in a cellculture with the a third leukocyte. After the second and thirdleukocytes have been cultured for a time sufficient to allowpro-tolerogenic allo-recognition, the supernatant of this cell culturecan be administered to the subject in need thereof. The supernatant canbe modified (e.g. filtered) to remove the second and/or third leukocyte.However, no specific size fractionation or enrichment of specificfractions is applied to the cell culture supernatant prior toadministering it to the subject. When the second and third leukocytesare cultured in the same medium (or in the same culture system), one ofthe two cell populations is inhibited/limited from proliferating (aslong as the other cell populations remains capable of proliferating).For example, the modified second leukocyte can be inhibited/limited fromproliferating prior to its co-culture with the third leukocyte.Alternatively, the third leukocyte can be inhibited/limited fromproliferating prior to its co-culture with the modified secondleukocyte. In the co-culture systems, it is important that at least oneof the two cell populations be able to proliferate and be consideredviable. In an optional embodiment, the second and/or third leukocyte canbe expanded and/or differentiated in vitro (from naïve to Treg) prior toor during co-culture.

As indicated above, in the cell culture system, the second leukocyte isallogeneic to the third leukocyte. In some embodiments, the secondleukocyte can be allogeneic to the subject and to third leukocyte.Alternatively, the second leukocyte can be xenogeneic to the subjectand/or to the third leukocyte. Optionally, one of the second or thirdleukocyte can be syngeneic or derived from the subject.

Usually, the cultured cellular preparation is obtained at least 24 hoursafter the initial contact between the second leukocyte and the thirdleukocyte. In some embodiment, the cultured cellular preparation isobtained at least 48 hours or at least 72 hours after the initialcontact between the second leukocyte and the third leukocyte. In anembodiment, the cultured cellular preparation can be obtained after atleast 24 hours of incubating a second leukocyte (for example graftedwith a 20 kDa PEG at a density of at 1.0 mM) with the third leukocyte.When the incubation takes place in a 24-well plate, the concentration ofeach leukocyte population can be at least 1×10⁶ cells.

In other embodiments, to provide the cellular preparations, aconditioned blood can be used. The conditioned blood can be obtained byadministering a first leukocyte, a first leukocyte population or a firstleukocytic relatively homogeneous population (e.g. all modified with thelow-immunogenic polymer and all refrained from proliferating) to thetest subject (usually an animal, such as a mouse). The blood isrecuperated from the test subject after a time sufficient to induce inthe transfused test subject a state of anergy or tolerance. It isimportant that the first leukocyte be administered to an immunecompetent test subject and that the blood or blood fraction be obtainedat a later a time sufficient to provide a conditioned blood. The testsubject is a subject being immune competent and having aTreg/pro-inflammatory T cell ratio which is substantially similar toage- and sex-matched healthy subjects. As used herein, the conditionedblood refers to physical components present in the blood and obtained byadministering the first leukocyte to the immune competent test subjectand having the pro-tolerogenic properties described herein. It isrecognized by those skilled in the art that the conditioned blood may beobtained more rapidly by increasing the amount of leukocytes beingadministered or administering more than once (for example one, twice orthrice) the modified leukocyte. Usually, the conditioned blood isobtained at least one day after the administration of the firstleukocyte. In some embodiment, the conditioned blood is obtained atleast 2, 3, 4, 5, 6, 7 or 8 days after the administration of the firstleukocyte. In an embodiment, the conditioned blood can be obtained byadministering at least 5×10⁶ polymer-modified leukocytes (for examplegrafted with at least 1.0 mM of 20 kDa PEG) to the test subject (e.g. amice) and recuperating the plasma five days later. In some embodiment,the conditioned blood can be obtained by administering at least 20×10⁶polymer-modified leukocytes. Methods for obtaining the blood or itsfractions (such as serum or plasma) are known to those in the art andusually involve centrifugation and cell lysis.

Once the cellular preparations have been obtained, they can beformulated for administration to the subject. The formulation step cancomprise admixing the cellular preparation, conditionedsupernatant/serum obtained (at a therapeutically effective dose) withpharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, and/or carriers. The formulations are preferably in aliquid injectable form and can include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces.The formulations can comprise pharmaceutically acceptable solubilizingagents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal,benzyl alcohol, parabens), bulking substances or tonicity modifiers(e.g., lactose, mannitol).

In addition, if the cellular preparations are destined to be used toprevent or limit an excessive immune reaction triggered by a vaccine,they can be formulated for administration with the vaccine. The cellularpreparation or the conditioned serum/supernatant can be formulated forsimultaneous administration with the vaccine by admixing the vaccinewith the cellular preparation or the conditioned serum/supernatant.Alternatively, the cellular preparation or the conditionedserum/supernatant can be formulated for administration prior to or afterthe vaccine, for example in a formulation that is physically distinctfrom the vaccine.

Further, if the cellular preparations are destined to be used to preventor limit an excessive immune reaction triggered by a transplant, theycan be formulated for administration prior to the transplantation. Thecellular preparations can be formulated for simultaneous administrationwith the transplant. Alternatively, the cellular preparations can beformulated for administration prior to or after the transplant.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I—Material and Methods

Human PBMC and Dendritic Cell Preparation.

Human whole blood was collected in heparinized vacutainer bloodcollection tubes (BD, Franklin Lakes, N.J.) from healthy volunteerdonors following informed consent. PBMC were isolated from diluted wholeblood using FicollePaque PREMIUM™ (GE Healthcare Bio-Sciences Corp,Piscataway, N.J.) as per the product instructions. The PBMC layer waswashed twice with 1× Hank's Balanced Salt Solution (HBSS; without CaCl₂and MgSO₄; Invitrogen by Life Technologies, Carlsbad, Calif.) andresuspended in the appropriate media as needed for mixed lymphocytereactions and flow cytometric analysis of Treg and Th17 phenotypes.Dendritic cells (DC) were prepared from isolated PBMC as described byO'Neill and Bhardwaj (O'Neill et al., 2005). Briefly, freshly isolatedPBMC were overlaid on Petri dishes for 3 h in AIM V serum free culturemedium (Invitrogen, Carlsbad, Calif.). Non-adherent cells were gentlywashed off the plate. The adherent cells (monocyte rich cells) weretreated with IL-4 and GM-CSF (50 and 100 ng/mL respectively; R&DSystems, Minneapolis, Minn.) in AIM V medium. Cells were again treatedwith IL-4 and GM-CSF on days 2 and 5. On day 6, cells were centrifugedand resuspended in fresh media supplemented with DC maturation factors(TNF-α, IL-1β, IL-6; R&D Systems, Minneapolis, Minn.) and prostaglandinE2 (Sigma Aldrich, St. Louis, Mo.). The mature DC-like cells wereharvested on day 7 and CD80, CD83, CD86 and HLA-DR expressions weredetermined to confirm DC maturation via flow cytometry (FACSCalibur™Flow Cytometer, BD Biosciences, San Jose, Calif.).

Murine Splenocyte and Tissue Harvesting.

All murine studies were done in accordance with the Canadian Council ofAnimal Care and the University of British Columbia Animal Care Committeeguidelines and were conducted within the Centre for Disease Modeling atthe University of British Columbia. Murine donor cells used for the invivo donation and in vitro studies were euthanized by CO₂. Threeallogeneic strains of mice were utilized for syngeneic and allogeneic invitro and in vivo challenge: Balb/c, H-2^(d); C57Bl/6, H-2^(b); and C3H,H-2^(k). Murine spleens, brachial lymph nodes, and peripheral blood werecollected at the indicated days. Mouse spleens and brachial lymph nodeswere dissected and placed into cold phosphate buffered saline (PBS; 1.9mM NaH₂PO₄, 8.1 mM Na₂HPO₄, and 154 mM NaCl, pH 7.3) containing 0.2%bovine serum albumin (BSA; Sigma Aldrich, St. Louis, Mo.) and kept onice until ready to process. Whole blood was collected in heparinizedtubes via cardiac puncture. Murine donor splenocytes were prepared fromfreshly harvested syngeneic or allogeneic spleens via homogenizationinto a cell suspension in PBS (0.2% BSA) using the frosted end of twomicroscope slides. The resultant cell suspension was spun down at 500×g.The splenocyte pellet was resuspended in 1 mL of 1× BD Pharm LYSE™lysing buffer (BD Biosciences, San Diego, Calif.) and incubated for 1min at room temperature. Lymph node cells were harvested via tissuehomogenization as described above, washed twice and resuspended in PBS(0.2% BSA) for flow cytometric analysis of Th17, Treg and murinehaplotype. Recipient peripheral blood lymphocytes were prepared vialysis of the red cells (BD Pharm Lyse lysing buffer; BD Biosciences, SanDiego, Calif.) at 1× concentration, followed by washing (1×) andresuspension in PBS (0.2% BSA) for flow analysis of Th17, Treg andmurine haplotype.

mPEG Modification (PEGylation) of PBMCs and Splenocytes.

Human PBMC and murine splenocytes were derivatized usingmethoxypoly(-ethylene glycol) succinimidyl valerate (mPEG-SVA; LaysanBio Inc. Arab, Ala.) with a molecular weight of 5 or 20 kDa aspreviously described (Scott et al., 1997; Murad et al, 1999A; Chen etal., 2003; Chen et al., 2006; Wang et al. 2011). Grafting concentrationsranged from 0 to 5.0 mM per 4×10⁶ cells/mL. Cells were incubated withthe activated mPEG for 60 min at room temperature in isotonic alkalinephosphate buffer (50 mM K₂HPO₄ and 105 mM NaCl; pH 8.0), then washedtwice with 25 mM HEPES/RPMI 1640 containing 0.01% human albumin. HumanPBMC were resuspended in AIM V media at a final cell density of 2.0×10⁶cells/mL for use in the MLR. Murine splenocytes used for in vivo studieswere resuspended in sterile saline at a final cell density of 2.0×10⁸cells/ml for i.v. injection. To determine if the simple presence of themPEG polymer itself altered the immune response either in vitro and invivo, additional studies were done with unactivated polymer incapable ofcovalent grafting to the cell surface. For these studies, allogeneichuman (in vitro studies) or syngeneic and allogeneic murine splenocytes(in vivo studies) were treated with non-covalently bound mPEG (solublemPEG) under the same reaction conditions described for the covalentgrafting studies. For clarity, “soluble mPEG” refers to cells treatedwith non-covalently grafted polymer while “mPEG-modified” refers totreatment with activated polymer resulting in the covalent grafting ofthe mPEG to the cell membrane.

In Vitro and In Vivo Cell Proliferation.

Cell proliferation (both in vitro and in vivo) was assessed via flowcytometry using the CELLTRACE™ CFSE (Carboxyfluorescein diacetate,succinimidyl ester) Cell Proliferation Kit (Invitrogen by LifeTechnologies e Molecular probes, Carlsbad, Calif.). Human and murinecells labeling was done according to the product insert at a finalconcentration of 2.5 mM CFSE per 2×10⁶ cells total. Donor and recipientcell proliferation was differentially determined by haplotype analysis.In some experiments, cell proliferation was measured by ³H-thymidineincorporation. In these experiments, donor splenocytes (5.12×10⁶ cellsper well) were co-incubated in triplicate in 96-well plates at 37° C.,5% CO₂ for 3 days. On day 3, all wells were pulsed with ³H-thymidine andincubated for 24 h at 37° C., 5% CO₂. Cellular DNA was collected onfilter mats using a Skatron cell harvester (Suffolk, U.K.) and cellularproliferation was measured by ³H-thymidine incorporation.

Mixed Lymphocyte Reaction (MLR)—Control and Conditioned Media.

The effects of polymer grafting (5 kDa SVAmPEG) on allorecognition invitro were assessed using two-way MLR (Murad et al, 1999A; Chen et al.,2003; Chen et al., 2006). PBMC from two MHC-disparate human donors werelabel with CFSE as described. Each MLR reaction well contained a totalof 1×10⁶ cells (single donor for resting or mitogen stimulation or equalnumbers for disparate donors for MLR). Cells were plated in multiwellflat-bottom 24-well tissue culture plates (BD Biosciences, DiscoveryLabware, Bedford, Mass.). PBMC proliferation, cytokine secretion, aswell as Treg and Th17 phenotyping was done at days 10 and 14. For flowcytometric analysis, the harvested cells were resuspended in PBS (0.1%BSA). While time course studies (Days 4, 7, 10 and 14) were done, thepresented studies show days 10 and 14. These extended studies are, infact, the most stringent test of the immunomodulatory effects of thegrafted polymer as membrane remodeling over this time could haveresulted in a latter onset of proliferation. To investigate in vitrowhether polymer grafting to allogeneic PBMC gave rise to tolerance oranergy, secondary)(2°) MLR studies were conducted using conditionedmedia. Conditioned media from a primary)(1°) 2 way-MLR was collected at72 h for conducting a secondary)(2°) MLR as schematically shown inFIG. 1. Conditioned media from the 1° MLR included: A1) restingunmodified PBMC; B2) resting PEGylated PBMC; C3) two-way MLR; and D4)two-way mPEG-MLR. The 2° MLR utilized freshly prepared MHC-disparatedonors (either the same as or different from) the initial plate andplated as described above. As shown in FIG. 1, the 2° MLR samplesincluded: A) resting PBMC; B) two-way MLR; P) mitogen stimulation; D)two-way mPEG-MLR. For these studies, PBMC were derivatized using 1 mM 5kDa SVAmPEG. Mitogen stimulation (PHA-P; Sigma-Aldrich, St. Louis, Mo.)of donor PBMC in the secondary plates was done to assess theproliferation potential and viability of cells incubated in theconditioned media. Human PBMC were challenged with 2 mg/ml per 1×10⁶cells of PHA-P. All plates were incubated at 37° C. (5% CO₂). Following13 days of incubation (37° C., 5% CO₂), the cell culture supernatantswere collected and cells were harvested from the 2° MLR plates. Cellproliferation was measured via CSFE-dilution of CD3⁺CD4⁺ lymphocytes byflow cytometry.

Immunophenotyping by Flow Cytometry.

The T lymphocytes populations (double positive for CD3⁺ and CD4⁺) inboth the in vitro and in vivo studies were measured by flow cytometryusing fluorescently labeled CD3 and CD4 monoclonal antibodies (BDPharmingen, San Diego, Calif.). Human and mouse Regulatory T lymphocytes(Treg) were CD3⁺/CD4⁺ and FoxP3⁺ (transcription factor) whileinflammatory Th17 lymphocytes cells were CD3⁺/CD4⁺ and IL-17⁺ (cytokine)as measured per the BD Treg/Th17 Phenotyping Kit (BD Pharmingen, SanDiego, Calif.). After the staining, the cells (1×10⁶ cells total) werewashed and resuspended in PBS (0.1% BSA) prior to flow acquisition.Isotype controls were also used to determine background fluorescence.All samples were acquired using the FACSCalibur™ flow cytometer (BDBiosciences, San Jose, Calif.) and CellQuest Pro™ software for bothacquisition and analysis.

Cytokine Quantitation.

Cell culture supernatants were collected from the 1° two-way MLR plateand stored at −80° C. prior to analysis. Conditioned media aliquots froma minimum of four independent experiments were used for quantificationof supernatant cytokine levels using the BD Cytometric Bead Array (CBA)system (BD Biosciences, San Diego, Calif.) for flow cytometry. The CBAsystem is a multiplexed bead based immunoassay used to quantitatemultiple cytokine levels in a single sample simultaneously byfluorescence-based emission and flow cytometry. Cytokine measuredincluded: IFNγ, TNF-α, IL-10, IL-5, IL-4, and IL-2 using the BD HumanTh1/Th2 Cytokine Kit I™. The IL-6 and IL-17A levels were measured usingthe BD CBA Human Soluble Protein Flex Set™. Both assays were performedfollowing the manufacturer's product instruction manual. Briefly, cellculture supernatants of resting unmodified PBMC, unmodified MLR,PEGylated (5 kDa SVAmPEG; one donor) resting PBMC, PEGylated MLR, andmitogen (PHA) stimulated PBMC were incubated at room temperature in thedark with a mixture of each cytokine antibody-conjugated capture beadand the PE-conjugated detection antibody. Following the incubation, thesamples were washed and acquired using a FACSCalibur™ flow cytometer andanalyzed using Cell-Quest Pro™ software. Cytokine protein levels weredetermined using the BD Cytometric Bead Array™ and FCAP Array™ analysissoftware (BD Biosicences, San Diego, Calif. and Soft Flow Inc, St. LouisPark, Minn.).

In Vivo Murine Studies.

To investigate whether mPEG grafting to leukocytes would have systemicin vivo effects, a murine adoptive transfer system was employed usingthree genetically different strains: Balb/c, H-2^(d); C57Bl/6, H-2^(b);and C3H, H-2^(k) (Chen et al., 2003; Chen et al., 2006). All mice(donors and recipients) were 9-11 weeks old. Donor splenocytes wereprepared and CSFE labeled as described. control and mPEG-grafted (1 mM,20 kDa SVAmPEG) syngeneic or allogeneic cells (20×10⁶ splenocytes) weretransfused intravenously (i.v.) via the tail vein into recipientanimals. BALB/c and C57BL/6 mice injected with sterile saline served ascontrol animals. Animals were euthanized by CO₂ at predeterminedintervals at which time blood, brachial lymph nodes and spleen werecollected and processed for Th17/Treg phenotyping analysis andsplenocyte proliferation studies by flow cytometry. Donor cellengraftment and proliferation were assessed via flow cytometry usingmurine haplotype (H-2K^(b) vs. H-2K^(d)) analysis. To determine thepersistence of the immunomodulation, mice were rechallenged (2°challenge) 30 days after the initial transfer of allogeneic ormPEGallogeneic splenocytes with unmodified allogeneic cells. At 5 dayspost 2° challenge, Treg and Th17 phenotyping of murine splenocytesisolated from the spleen, lymph node and peripheral blood was againassessed via flow cytometry.

Statistical Analysis.

Data analysis was conducted using SPSS™ (v12) statistical software(Statistical Products and Services Solutions, Chicago, Ill., USA). Forsignificance, a minimum p value of <0.05 was used. For comparison ofthree or more means, a one-way analysis of variance (ANOVA) wasperformed. When significant differences were found, a post-hoc Tukeytest was used for pair-wise comparison of means. When only two meanswere compared, student-t tests were performed.

Example II—In Vitro and In Vivo Immunomodulation

The material and methods used in this example are provided in Example I.

To determine the effects of polymer modification on the immune response,initial in vitro experiments examined the cytokine burst characterizingcontrol and polymer modified MLR. The polymer-mediated immune modulationof human PBMC resulted in significant changes in the cytokine profile ofthe conditioned media obtained from the 1° MLR plate (FIGS. 1 and 2). Asshown in FIG. 2, control MLRs yielded elevated concentrations of IL-2,IFN-γ, IL-17A, TNF-α and IL-6 relative to resting unmodified orPEGylated PBMC. In contrast to the control MLR, the mPEG-MLR (one donorpopulation PEGylated with 1 mM 5 kDa SVA-mPEG) resulted in the virtuallycomplete inhibition (p<0.001) of secretion for the proinflammatorycytokines examined. However, IL-10 was preferentially elevated in themPEG-MLR. In the conditioned media, IL-10 levels were 2.01±1.26,8.90±2.10, 1.69±0.64 and 1.33±0.73 ng/ml for the resting PBMC, mPEG-MLR,resting mPEG-PBMC and Control MLR, respectively. As noted, in themPEG-MLR, IL-10 levels were significantly (p<0.01) increased suggestingthe induction of an immunosuppressive state. Importantly, this cytokinequiescent state was not due to loss of cell viability as evidenced bythe very low levels of non-viable cells detected following 72 hincubation (FIG. 2).

The conditioned media produced from the initial 72 h MLR exerted asignificant effect on the 2° MLR as demonstrated in FIG. 3. While the 1°media from resting PBMC showed no significant effect on the 2° MLR, themedia from the 1° Control MLR demonstrated a significant (p<0.01)pro-proliferative effect in the 2° MLR. As shown, the mean proliferationindex of the 2° MLR increased from 26.05±12.47 to 44.72±17.13 in thepresence of conditioned media from the 1° Control MLR. Thepro-inflammatory effect of the 1° MLR media was noted on even theresting PBMC and PHA-stimulated cells. In contrast, the 1° conditionedmedia from the mPEG-MLR demonstrated a significant (p<0.001)anti-proliferative effect in not only the 2° MLR but also thePHA-stimulated cells. The differential proliferation response betweenthe control and mPEG-MLR conditions for matching experiments is noted bythe lines connecting paired experiments. While not shown, soluble mPEG(5 kDa) had no effect on cytokine levels in the 1° conditioned media noron the proliferation of PBMC mediated by allorecognition (control MLR)or by mitogen (PHA) stimulation.

Furthermore, as shown in FIG. 4, the proliferation index was positivelycorrelated with an increased population of Th17 T cells and inverselycorrelated with Treg lymphocytes numbers. As demonstrated, the 1°conditioned media from the control MLR yielded elevated levels of Th17cells and decreased levels of Treg lymphocytes. In comparison, the 1°media from the mPEG-MLR resulted in significantly elevated (p<0.001)levels of Treg cells and a virtually non-existent population of Th17lymphocytes. The source of the conditioned media also impacted theefficacy of PHA stimulation. As shown, conditioned media from themPEG-MLR significantly inhibited mitogen proliferation while the controlMLR conditioned media significantly enhanced proliferation relative toboth media from resting PBMC (p<0.01) and resting mPEG-PBMC (p<0.001).

Hence, the in vitro experiments demonstrated that covalent grafting mPEGto human PBMC resulted in an immunomodulatory effect governed in part bychanges in the Th17 and Treg populations. Moreover, these conditionedmedia experiments demonstrated that this immunomodulatory effect arisesfrom soluble factors that might be able to induce a systemic effect invivo. To determine if similar effects would be observed in vivo, amurine splenocyte adoptive transfer model was utilized. As demonstratedin FIG. 5, PEGylated donor splenocytes resulted in a significant in vivoimmunomodulatory effect giving rise to elevated levels of Treglymphocytes within the spleen, brachial lymph node, and peripheralblood. As noted, in all three tissues, a significant (p<0.001 at 120 h)time-dependent increase in Treg lymphocytes over that observed in naïvemice was noted in mice receiving mPEG-modified allogeneic donor cells.In stark contrast, a significant (p<0.001) decrease in Tregs 48 hpost-injection relative to naïve mice) is noted in mice transfused withunmodified allogeneic splenocytes. The absolute difference between theunmodified (control) and PEGylated splenocytes, shown by the stippledarea, demonstrates the true magnitude of the differential impact ofdonor cell PEGylation. Importantly, as noted at 120 h, transfusion ofsoluble mPEG, syngeneic cells or mPEG-syngeneic cells had no significanteffect on the Treg lymphocyte population.

As foreshadowed by our in vitro human PBMC findings (Example II), murineTh17 lymphocyte levels were influenced by the PEGylation state of theallogeneic donor cells (FIG. 6). While unmodified allogeneic murinedonor cells resulted in a significant (p<0.001), time-dependent,increase in the Th17 cell population in the spleen, brachial lymph nodeand peripheral blood, the covalent grafting of mPEG to the membrane ofthe donor splenocytes resulted in the complete abrogation of theincrease. Indeed, the Th17 population remained at resting levels. Theabsolute difference between the unmodified and mPEG-modified donor cellsis denoted by the stippled area. As with the Treg population,transfusion of soluble mPEG, syngeneic cells or mPEG-syngeneic cells hadno significant effect on the Th17 lymphocyte population at 120 h.

As also shown on FIG. 7, normal mice have significantly higher levels ofTregs (Spleen ˜10% total CD4+ T cells) relative to Th17 T Cells (Spleen˜0.05% total CD4+ T cells). Further, treatment with unmodifiedallogeneic cells results in production of Th17 cells and loss of Tregs.In contrast, polymer modified allogeneic cells maintain (even increase)Tregs and prevents Th17 production.

As might be anticipated, the mPEG-allogeneic splenocyte mediatedincrease in Treg cells in the peripheral blood samples occurred later inthe studied time course (96 h) compared to either of the lymphatictissues (spleen and lymph nodes; 48 h). This clearly suggests that Tcell proliferation initially occurred within the lymphatic tissues andsecondarily migrated into the peripheral blood. A similar timedependency was noted with the Th17 proliferation induced by theunmodified splenocyte populations. Proliferation initially occurredwithin lymphatic tissue within ˜48 h and only appeared within theperipheral blood after ˜96 h.

Of importance was the observation that the immunomodulatory effects ofthe PEGylated splenocytes were long lived and prevented subsequentchanges in Treg and Th17 levels consequent to rechallenge withunmodified allogeneic cells. As shown in FIG. 8, 30 days posttransfusion with polymer modified splenocytes, Treg levels remainsignificantly elevated and are similar to levels recorded at 120 h postchallenge. In contrast, Th17 levels remained similar to or decreasedfrom that observed in naïve mice at day 30. Of even more interest, asecondary adoptive transfer of unmodified allogeneic splenocytes (30days post 1° challenge; measured at 120 h) to mice previously treatedwith PEGylated allogeneic showed no significant decrease in Treg cells,or increase in Th17 cells, relative to the day 30 levels. This was indirect contrast to that observed in naïve mice (FIG. 5) injected withunmodified allogeneic cells that demonstrated a dramatic decrease inTreg lymphocytes. Indeed, Treg levels remain significantly elevatedabove that seen in naïve mice and very similar to those levels observedat 5 days post PEGylated splenocytes transfusion.

To determine if the observed in vivo murine findings gave rise to atolerance to a specific H-2 haplotype or a more generalized anergy toallogeneic tissues, in vitro two-way murine MLR studies of threeallogeneic splenocyte populations (Balb/c, H-2^(d); C57Bl/6, H-2^(b);and C3H, H-2) were done. As demonstrated in FIG. 9, the immunomodulationarising following exposure to polymer-grafted H-2 disparate splenocytesis not specific to the haplotype of the mPEGmodified spelenocytesthereby suggestive of an anergic state. As shown, PEGylation ofstimulator (i.e., irradiated and incapable of proliferation) splenocytesvery effectively attenuated allorecognition and proliferation of theresponder cell population within a one-way MLR. Moreover, forcomparative purposes, the anti-proliferative dose-response effect ofcyclosporine A (CSA; which induces a pharmacologically-induced anergy)in a one-way murine MLR under the same experimental condition is shown.Interestingly, the type of polymer-modified cell is important. Humanlymphocytes and murine splenocytes express high levels of“self-antigens” (Human Leukocyte Antigens (HLA) and mouse H-2 proteins).If cells devoid of these highly immunogenic antigens are used in themurine model, no changes in either Tregs or Th17 cells are observed. Inmice injected with unmodified allogeneic erythrocytes, Treg levelswithin the spleen, lymph node and peripheral blood were (respectively):91.7%, 95.0% and 107.0% of control mouse values. Similarly unchanged,Th17 levels were (respectively): 71.2%, 112.1% and 79.2% of controlmouse values. Thus, allogeneic murine RBC do not elicit any significantchanges in the systemic levels of either Treg or Th17 lymphocytes. Thisfinding was observed despite some antigenic differences between the RBCin H-2 disparate mice. In support of the low immunogenicity of thesegenetically different RBC, allogeneic RBC exhibit normal in vivocirculation nor do they elicit a significant immune response. Hence,polymer coupled to a low-immunogenicity allogeneic cell can not inducethe immunomodulation noted with the highly immunogenic splenocytes.

Bioengineering of donor cells and/or tissues may provide significantopportunities to attenuate both the recognition and rejection ofallogeneic tissues. One polymer-mediated approach to this end is thepolymer modification of donor cells via the covalent grafting of mPEG(or other low immunogenicity polymers) to the exterior face of cellmembranes. Consequent to membrane derivatization, mPEG-modifiedallogeneic and xenogeneic cells demonstrated a global, multivalent,attenuation of antigenicity and immunogenicity. This effect arouse inpart from charge camouflage and significantly diminished cell:cell(e.g., T cell:APC or T cell:islet cell) and ligand:receptor (e.g.,antibody:antigen or CD28:CD80) interactions. The efficacy was dependenton polymer molecular weight (i.e., size) and grafting density.

However, the inhibition of cell:cell and ligand:receptor interactionsare a ‘local’ immunomodulatory event arising from the steric and chargecamouflaging effects of the grafted polymer. For the induction oftolerance, a systemic and persistent immunomodulatory effect would benecessary. As demonstrated herein, covalent grafting of mPEG toallogeneic lymphocytes (human PBMC or murine splenocytes) dramaticallyreduced allorecognition at both the local (cell:cell; MLR) and systemic(in vivo murine models) levels. Importantly, as demonstrated in our invivo studies, it is not the donor cells that differentiate into Treg orTh17 cells, rather it is the recipients immune system that responds tothe control or PEGylated splenocytes and upregulates production ofeither Th17 (upon challenge with unmodified splenocytes) or Treg (uponchallenge with mPEG-splenocytes) populations. This was noted by both theabsence of CFSE-staining (only donor cells were stained) and H-2phenotyping of the Th17 and Treg cell populations.

The observed immunosuppressive state induced by PEGylated lymphocytes issurprisingly long lasting in vivo. As noted in FIG. 8, the elevatedlevels of Treg lymphocytes noted at day 5 persist to day 30. Moreover,the presence of these Treg (as well as other probable immunologicalevents) prevents a pro-inflammatory response to unmodified allogeneicsplenocytes administered at day 25. Indeed, no increase in Th17lymphocytes is noted in the immunomodulated mice. Moreover, for thesystemic tolerance/anergy to occur, the polymer must be grafted to ahighly immunogenic cell type (e.g., lymphocyte and/or antigen presentingcells) as less immunogenic cells, such as H-2 disparate erythrocytes, donot alter the immune (Treg/Th17) response. While allogeneic murineerythrocytes do express antigenic differences at the membrane, thesecells are only weakly immunogenic eliciting weak IgG responses andtypically remaining in the vascular circulation with a near normalhalf-life. Also of critical importance, induction of both local andsystemic immunomodulation requires the covalent grafting of the polymerto the cell, as soluble mPEG±allogeneic cells has no effect on thepopulation dynamics of either Treg or Th17 lymphocytes in vitro or invivo.

The balance between Treg and Th17 cells has been identified as a keyfactor that orchestrates the tolerance/inflammation level of humanimmune system. Regulatory T cells provide suppressor effect and maintaintolerance, while Th17 cells mediate and are indicative of apro-inflammatory state. Hence, the polymer-mediated modulation of thisbalance may be clinically useful. Recent findings have shown thatcyclosporine, a clinically used immunosuppressive agent, has substantialeffects on the Treg/Th17 cell response; though this may be mediated byTh17 cytotoxicity as Treg cells cultured in the presence of rapamycin,but not cyclosporine A, are found to suppress ongoing alloimmuneresponses. Additionally, mycophenolic acid, another immunosuppressiveagent, was found to shift the lymphocyte polarization by inhibitingIL-17 expression in activated PBMC in vitro. Of clinical importance, allof these pharmacologic agents exert significant systemic toxicity andtheir ongoing use requires substantial monitoring.

As evidenced by these results, induction of tolerance or anergy intransfusion and transplantation medicine by the polymer modification ofallogeneic leukocytes may provide a less toxic approach than currentconventional pharmacologic agents. Current efforts to prevent and/orregulate the consequences of allorecognition involve phenotype matching(ranging from blood group to HLA matching) and the use ofimmunosuppressive agents (FIG. 10A). While extensive tissue matching(e.g., blood groups, HLA) can dramatically enhance transfusion ortransplantation success, the necessity of tissue matching dramaticallyreduces the potential pool of donor tissues. Even in a tissue asplentiful as blood, extensive non-ABO matching for chronicallytransfused patients (e.g., sickle cell disease), while considereddesirable, is costly and often difficult to achieve due to the scarcityof appropriately matched donor cells. This difficulty is greatlyexaggerated with less common tissues and organs (e.g., islets andkidneys).

Thus, pharmacological interventions have been employed to enhance theprobability of successful donor tissue engraftment (FIG. 10B). The datapresented here suggests that polymer encapsulation “of”, or grafting“to”, donor tissue may be further enhanced or replaced by a tolerogeneicor anergic approach. Rather, the prechallenge of a potential tissuerecipient with PEGylated donor specific (or simply allogeneic; see FIG.9) PBMC several (˜5) days prior to tissue transplantation could be usedto induce a tolerogenic state within the recipient as shown in FIG. 10B.Elevated levels of Tregs and the down-regulation of Th17 cells woulddiminish the risks of both hyper-acute and acute rejection of the donortissue. There are several substantial advantages for this approach.Primary amongst these are the easy collection of donor specific (orsimply allogeneic) PBMC, the ease of PEGylation of the PBMC as well asthe ease of administration to the transplant recipient. While apotential risk of lymphocyte transfusions is transfusion associatedgraft versus host disease (TA-GVHD) in immunosuppressed patients, it waspreviously demonstrated that PEGylation effectively blocks TA-GVHD in amurine model (Chen et al., 2003; Chen et al., 2006). Moreover, thisprocess could be used in conjunction with irradiated PBMC thus obviatingany risk of TAGVHD. Irradiated cells retain their allo-stimulatoryeffects and PEGylation similarly inhibits this allorecognition andproliferation.

In summary, polymer modification of allogeneic donor lymphocytesprevents allorecognition at the cell:cell level and also gives rise tosystemic immunomodulation. The systemic immunomodulation is evidenced bya significant up-regulation of Treg cells and a significantdown-regulation of pro-inflammatory Th17 cells. This immunomodulation ispersistent (˜30 days) and prevents subsequent pro-inflammatory responsesto unmodified allogeneic cells. The polymer effect is dependent upon itscovalent grafting to allogeneic cells as soluble PEG itself has noimmunomodulatory effects. The clinical use of PEGylated (or othercovalently grafted polymers) allogeneic lymphocytes to pre-challengetissue recipients 5 days or more to transplantation may be useful ininducing a tolerogenic state and preventing acute rejection and/orenhancing tissue engraftment.

Example III—In Vivo Immunomodulation in Nod Mice

Some of the material and methods referred to in this example areprovided in Example I.

In the NOD mice, autoimmune destruction of the pancreatic islets occurswithin approximately 16 weeks and was confirmed with elevated bloodglucose measures. The lymphocytes from pre-diabetic and diabetic animalshas been obtained from the spleen, the brachial lymph node and thepancreatic lymph node. These lymphocytes have been submitted to flowcytometry using anti-IL-17A (PE) and anti-FoxP3 (Alexa 697) antibodies.As shown in FIG. 11, significant changes in the levels of Th17 and Treglymphocytes are noted in the spleen, brachial lymph node and pancreaticlymph nodes upon conversion of NOD mice from non-diabetic to diabeticstate. These changes are characterized by dramatically increased Th17(top numbers in each panels) and significantly decreased Treg (lowernumbers in each panels) lymphocytes. Tregs: *, p<0.001 from non-diabeticNOD mice. Th17: ** p<0.001 from non-diabetic NOD mice.

The NOD mice (8 to 10 week-old) have been treated with allogeneicleukocytes (as described in Example I) and mPEG-allogeneic leukocyte (asdescribed in Example I) and were compared to untreated control mice(naïve or NOD in Table 1). Th17 levels have been measured in varioustissues (as described in Example I). Peripheral blood samples of thegroups were pooled for analysis, all other samples were measuredindividually. Five male NOD mice per group were used. The results areshown in Table 1 provided below.

TABLE 1 Treatment of NOD mice with unmodified or mPEG-modifiedallogeneic cells. Unmodified cells results in a potent inflammatorystate as showby increased Th17 cells. In contrast, administration ofmPEG-allogeneic cells does not induce inflammation. Th17 Tissue NODAllogeneic mPEG Allogeneic Blood 0.38 0.67 0.17* Spleen 0.10 ± 0.01 2.32± 0.38 0.11 ± 0.01* Brachial L. Node 0.08 ± 0.01 1.25 ± 0.35 0.06 ±0.01* Pancreatic L. Node 0.05 ± 0.01 0.27 ± 0.08 0.07 ± 0.01* *p < 0.001relative to unmodified allogeneic cell treated.

Example IV—POZ Polymer for Inducing Tolerance or Anergy

Some of the material and methods referred to in this example areprovided in Example I.

Human PBMC and Dendritic Cell Preparation.

Human whole blood was collected in heparinized vacutainer bloodcollection tubes (BD, Franklin Lakes, N.J.) from healthy volunteerdonors following informed consent. PBMC were isolated from diluted wholeblood using FicollePaque PREMIUM™ (GE Healthcare Bio-Sciences Corp,Piscataway, N.J.) as per the product instructions. The PBMC layer waswashed twice with 1× Hank's Balanced Salt Solution (HBSS; without CaCl₂and MgSO₄; Invitrogen by Life Technologies, Carlsbad, Calif.) andresuspended in the appropriate media as needed for mixed lymphocytereactions and flow cytometric analysis of Treg and Th17 phenotypes.Dendritic cells (DC) were prepared from isolated PBMC as described byO'Neill and Bhardwaj (O'Neill et al., 2005). Briefly, freshly isolatedPBMC were overlaid on Petri dishes for 3 h of in AIM V serum freeculture medium (Invitrogen, Carlsbad, Calif.). Non-adherent cells weregently washed off the plate. The adherent cells (monocyte rich cells)were treated with IL-4 and GM-CSF (50 and 100 ng/mL respectively; R&DSystems, Minneapolis, Minn.) in AIM V medium. Cells were again treatedwith IL-4 and GM-CSF on days 2 and 5. On day 6, cells were centrifugedand resuspended in fresh media supplemented with DC maturation factors(TNF-α, IL-1b, IL-6; R&D Systems, Minneapolis, Minn.) and prostaglandinE2 (Sigma-Aldrich, St. Louis, Mo.). The mature DC-like cells wereharvested on day 7 and CD80, CD83, CD86 and HLA-DR expressions weredetermined to confirm DC maturation via flow cytometry (FACSCalibur™Flow Cytometer, BD Biosciences, San Jose, Calif.).

mPEG Modification (PEGylation) of PBMCs and Splenocytes.

Human PBMC and murine splenocytes were derivitized usingmethoxypoly(-ethylene glycol) succinimidyl valerate (mPEG-SVA; LaysanBio Inc. Arab, Ala.) with a molecular weight of 20 kDa as described inExample I. Grafting concentrations ranged from 0 to 3.0 mM per 4×10⁶cells/mL.

POZ Modification (POZylation) of PBMCs and Splenocytes.

N-hydoxysuccinimidyl ester of polyethyloxazoline propionic acid(SPA-PEOZ; Serina Therapeutics, Huntsville, Ala.) with a molecularweight of 20 kDa were grafted on the cells as described in Example I.Grafting concentrations ranged from 0 to 3.0 mM per 4×10⁶ cells/mL.

In Vitro and In Vivo Cell Proliferation.

Cell proliferation (both in vitro and in vivo) was assessed via flowcytometry using the CellTrace™ CFSE (Carboxyfluorescein diacetate,succinimidyl ester) Cell Proliferation Kit (Invitrogen by LifeTechnologies e Molecular probes, Carlsbad, Calif.) as described inExample I.

Mixed Lymphocyte Reaction (MLR)—Control and Conditioned Media.

The effects of polymer grafting (20 kDa SVAmPEG or 20 kDa POZ) onallorecognition in vitro were assessed using two-way MLR (Murad et al,1999A; Chen et al., 2003; Chen et al., 2006) as described in Example I.

A 2-way MLR was conducted using either PEGylated or POZylated humancells. As shown on FIG. 12, the grafting of equimolar concentrations ofwither 20 kDa mPEG or PEOZ (POZ) on a human mixed lymphocyte reaction(MLR) had similar effects on cellular proliferation.

V—In Vivo Modulation of TREG:Th17 Ratio by Polymer-Modified Lymphocytes

Some of the material and methods referred to in this example areprovided in Example I.

Non-modified allogeneic splenocytes (20×10⁶) and mPEG-modifiedallogeneic splenocytes (20×10⁶) have been intravenously administered tomouse (naïve 8-week old Balb/c mouse; 10 mice per treatment condition).After 5 days, the spleen and the lymph nodes were harvested and theCD4-positive cells they contained were further analyzed by flowcytometry. As shown in FIGS. 13A (annexin V staining) and 13B(mitochondrial depolarization), the administration of mPEG-modifiedallogeneic splenocytes, when compared to the administration ofnon-modified allogeneic splenocytes, increased the number of apoptoticCD4-positive cells. As shown in FIG. 13C, the administration ofmPEG-modified allogeneic splenocytes, when compared to non-modifiedallogeneic splenocytes, also increased the intracellular expression ofIL-10 in CD4-positive cells. Further, the administration of non-modifiedallogeneic splenocytes caused a mean decrease in mouse weight whereasthe administration of mPEG-modified allogeneic splenocytes caused a meanincrease in mouse weight (FIG. 13D).

Non-modified allogeneic splenocytes (either 5, 20 or 40×10⁶ C57BL/6cells) and mPEG-modified allogeneic splenocytes (either 5, 20 or 40×10⁶C57BL/6 cells grafted at a density of 0.5 mM, 1 mM or 4 mM) have beenintravenously administered to mouse (5 Balb/c mice/treatment condition).After 5 days, the spleen and the lymph nodes were harvested and theCD4-positive cells they contained were further analyzed by flowcytometry. As shown in FIG. 14, the administration of mPEG-modifiedallogeneic splenocytes increased the percentage of Treg cells anddecreased the percentage of Th17 cells. As also shown in FIG. 14, theadministration of non-modified allogeneic splenocytes decreased thepercentage of Treg cells and increased the percentage of Th17 cells.Surprisingly, the increase in Treg cell counts observed after theadministration of mPEG-modified allogeneic splenocytes occurred withoutan increase in spleen weight while the increase in Th17 cell countsobserved after the administration of the non-modified allogeneicsplenocytes correlated with an increase in spleen weight (a mean 1.5×increase, data not shown).

Saline, syngeneic splenocytes, non-modified allogeneic splenocytes(20×10⁶ C57BL/6 cells) and mPEG-modified allogeneic splenocytes (20×10⁶C57BL/6 cells grafted at a density of 1 mM PEG) have been intravenouslyadministered to mouse either once (at day 0, e.g. condition 1) or thrice(at days 0, 2 and 4, e.g. condition 3) (20×10⁶ C57BL/6 cells grafted ata density of 1 mM PEG). After 5 or 10 days, the spleen and lymph nodeswere harvested and the CD4-positive cells they contained were furtheranalyzed by flow cytometry with an anti-CD279 antibody. As shown inFIGS. 15A and B, the administration of mPEG-modified allogeneicsplenocytes increased the number of CD279-positive cells (with respectto the total number of CD4-positive cells), in the spleen and in thelymph nodes, when compared to mock-treated or syngeneic-treated animals.As also shown in FIGS. 15A and B, the administration of non-modifiedallogeneic splenocytes decreased the number of CD279-positive cells(with respect to the total number of CD4-positive cells), in the spleenand in the lymph nodes, when compared to mock-treated orsyngeneic-treated animals. Ten days after the administration ofmPEG-modified allogeneic splenocytes a decrease the percentage of NKcells was observed in both the spleen and the brachial lymph node (FIG.16). Further, the administration of non-modified allogeneic splenocyteswas also shown to increase the percentage of NK cells in both the spleenand the brachial lymph node (FIG. 16). Further, as shown in Table 2below, the administration of mPEG-allogeneic splenocyte attenuated NKCell alloresponse and baseline levels in recipient mice (as measured byflow cytometry using a NK1.1 antibody.

TABLE 2 Percentage of NK1.1-positive cells in mice having receivedsaline, syngeneic splenocytes, non-modified allogeneic splenocytes andmPEG-modified allogeneic splenocytes. Cells were harvested 10 days afterthe last injection Type of cells administered Percentage of NK1.1- (20 ×10⁶ cells) Number of doses positive cells None (saline) 1 1.12 None(saline) 3 0.97 Syngeneic 1 0.94 Syngeneic 3 0.91 Non-modifiedallogeneic 1 2.26 Non-modified allogeneic 3 2.30 mPEG-modifiedallogeneic 1 0.29 mPEG-modified allogeneic 3 0.21

The thymus of these animals has also been harvested and the thymic cellscharacterized. As shown in FIG. 17A, the administration of mPEG-modifiedallogeneic splenocytes increased microchimerism in the thymus ofrecipient animals as shown by the number of CFSE labeled allogeneicdonor cells in the thymus. Under normal conditions only 6 to 10% of theinjected donor CD4-positive splenocytes are Treg (17A; open barsegment). But as shown in FIG. 17B, the administration of mPEG-modifiedsplenocytes increased the total percentage in thymic Treg cells (donor,open bar; recipient grey bar) in the recipient. In contrast, theadministration of non-modified allogeneic splenocytes decreased the invivo thymic Treg cells. Further, the administration of non-modifiedallogeneic splenocytes increased the percentage of thymic Th17 cells,while the administration of the mPEG-modified allogeneic splenocytesdecreased the percentage of thymic Th17cells (FIG. 17C).

VI—In Vivo Modulation of Treg:Th17 Ratio by Conditioned Media ObtainedVia Polymer-Modified Lymphocytes

Some of the material and methods referred to in this example areprovided in Example I.

Conditioned Serum.

Conditioned serum was obtained (by bleeding the animal and separatingthe cellular components of blood from the serum via centrifugation) fivedays after mice (Balb/c; N=5) received saline, unmodified syngeneicsplenocytes (Balb/c), unmodified allogeneic splenocytes (20×10⁶ C57BL/6cells) or mPEG-modified allogeneic splenocytes (20×10⁶ C57BL/6 cellsgrafted at a density of 1 mM PEG). The serum from naïve animals was alsoobtained as a control. The conditioned or naïve serum (100 μl) was thenadministered (i.v. tail vein injection) once (at day 0) or thrice (atdays 0, 2 and 4) to recipient mice (Balb/c; N=5). Five days after thelast administration, a blood sample, the spleen and the brachial lymphnodes were obtained from the treated animals and the leukocytes theycontained were analyzed.

As shown on FIG. 18, the administration of the conditioned serum fromanimals having received unmodified allogeneic splenocytes caused in vivoa reduction in the levels of Tregs, while increasing the levels of Th17cells in both the spleen and the lymph nodes. As also shown on FIG. 18,the administration of the conditioned serum from animals having receivedpolymer modified allogeneic splenocytes caused in vivo an increase inthe levels of Tregs as well as a decrease in the levels of Th17 cells,both in the spleen and the lymph node.

This modulation in Treg/Th17 ratio was also shown to be associated inthe long term modification of the expression of pro-/anti-inflammatorycytokine positive CD4+ lymphocytes. As shown on FIG. 19, theadministration of the conditioned serum from animals having receivedunmodified allogeneic splenocytes caused in vivo an increase in theexpression of pro-inflammatory cytokines (IL-2, TNF-α, IFN-γ and IL-4)positive lymphocytes while the administration of the conditioned serumfrom animals having received polymer modified allogeneic splenocytescaused in vivo an increase in the expression of anti-inflammatorycytokines (IL-10) in CD4+ lymphocytes. These results were observed forat least 30 days and 60 days after the last administration. Similarobservations have been observed 270 days after the last administration(data not shown).

The administration of the conditioned medium also caused a shift in theTreg subsets. As shown on FIG. 20, the administration of the conditionedserum from animals having received unmodified allogeneic splenocytescaused in vivo decrease in all Treg subsets (Foxp3⁺, CD25⁺ and CD69⁺) inthe spleen and the lymph nodes. The administration of the conditionedserum from animals having received polymer modified allogeneicsplenocytes caused in vivo an increase all Treg subsets. SurprisinglyCD69⁺ Treg cells demonstrated the most significant increase relative tonaïve mice.

As shown on FIG. 21, the administration of the conditioned serum fromanimals having received unmodified allogeneic splenocytes caused in vivoa reduction in the levels of Tregs, while increasing the levels of Th17cells in the spleen, the lymph nodes and the blood. As also shown onFIG. 21, the administration of the conditioned serum from animals havingreceived polymer modified allogeneic splenocytes caused in vivo anincrease in the levels of Tregs as well as a decrease in the levels ofTh17 cells, in the spleen, the lymph node and the blood.

REFERENCES

-   Bradley A J, Test S T, Murad K L, Mitsuyoshi J, Scott M D.    Interactions of IgM ABO antibodies and complement with    methoxy-PEG-modified human RBCS. Transfusion 2001; 41:1225-33.-   Bradley A J, Scott M D. Immune complex binding by immunocamouflaged    [poly(ethylene glycol)-grafted] erythrocytes. Am J Hematol 2007;    82:970-5.-   Chen A M, Scott M D. Current and future applications of    immunological attenuation via pegylation of cells and tissue.    BioDrugs 2001; 15:833-47.-   Chen A M, Scott M D. Immunocamouflage: prevention of    transfusion-induced graft-versus-host disease via polymer grafting    of donor cells. J Biomed Mater Res A 2003; 67:626-36.-   Chen A M, Scott M D. Comparative analysis of polymer and linker    chemistries on the efficacy of immunocamouflage of murine    leukocytes. Artif Cells Blood Substit Immobil Biotechnol 2006;    34:305-22.-   Le Y, Scott M D. Immunocamouflage: the biophysical basis of    immunoprotection by grafted methoxypoly(ethylene glycol) [mpeg].    Acta Biomater 2010; 6:2631-41.-   McCoy L L, Scott M D. Broad spectrum antiviral prophylaxis:    inhibition of viral infection by polymer grafting with    methoxypoly(ethylene glycol). In: PF T, editor. Antiviral drug    discovery for emerging diseases and bioterrorism threats. Hoboken,    N.J.: Wiley & Sons; 2005. p. 379-95.-   Murad K L, Gosselin E J, Eaton J W, Scott M D. Stealth cells:    prevention of major histocompatibility complex class II-mediated    T-cell activation by cell surface modification. Blood 1999A;    94:2135-41.-   Murad K L, Mahany K L, Brugnara C, Kuypers F A, Eaton J W, Scott    M D. Structural and functional consequences of antigenic modulation    of red blood cells with methoxypoly(ethylene glycol). Blood 1999B;    93:2121-7.-   O'Neill D W, Bhardwaj N. Differentiation of peripheral blood    monocytes into dendritic cells. Curr Protoc Immunol; 2005. Chapter    22: Unit 22F.4.-   Scott M D, Murad K L, Koumpouras F, Talbot M, Eaton J W. Chemical    camouflage of antigenic determinants: stealth erythrocytes. Proc    Natl Acad Sci USA 1997; 94:7566-71.-   Sutton T C, Scott M D. The effect of grafted methoxypoly(ethylene    glycol) chain length on the inhibition of respiratory syncytial    virus (RSV) infection and proliferation. Biomaterials 2010;    31:4223-30.-   Wang D, Toyofuku W M, Chen A M, Scott M D. Induction of    immunotolerance via mPEG grafting to allogeneic leukocytes.    Biomaterials. 2011 December; 32(35):9494-503.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A method of increasing a ratio of the level ofendogenous regulatory T (Treg) cells to the level of endogenouspro-inflammatory T cells in a subject in need thereof for treating oralleviating the symptoms associated with an auto-immune diseaseafflicting the subject, said method comprising administering to thesubject a therapeutic amount of: a first cellular preparation comprisinga first leukocyte having a cytoplasmic membrane associated to alow-immunogenic biocompatible polymer, wherein the low-immunogenicbiocompatible polymer is covalently associated with the first leukocyte,and wherein the first leukocyte is allogeneic to the subject and isirradiated; wherein the low-immunogenic biocompatible polymer ispolyethylene glycol (PEG), or 2-alkyloxazoline (POZ), or hyperbranchedpolyglycerol (HPG); thereby increasing the ratio of the level of Tregcells to the level of pro-inflammatory T cells in the subject fortreating or alleviating the symptoms associated with an auto-immunedisease afflicting the subject.
 2. The method of claim 1, wherein thefirst leukocyte is a T cell.
 3. The method of claim 2, wherein the Tcell is a CD4-positive T cell or a CD8-positive T cell.
 4. The method ofclaim 1, wherein the auto-immune disease is at least one of type Idiabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, lupus,immune thrombocytopenia, experimental autoimmune encephalomyelitis,autoimmune uveitis, inflammatory bowel disease, scleroderma and Crohn'sdisease.
 5. The method of claim 1, wherein the low-immunogenicbiocompatible polymer is polyethylene glycol (PEG).
 6. The method ofclaim 1, wherein the first leukocyte is a peripheral blood mononuclearcell.
 7. The method of claim 1, wherein the first leukocyte is asplenocyte.
 8. A method of increasing a ratio of the level of endogenousregulatory T (Treg) cells to the level of endogenous pro-inflammatory Tcells in a subject in need thereof for treating or alleviating thesymptoms associated with an auto-immune disease afflicting the subject,said method comprising: (i) providing a first leukocyte having acytoplasmic membrane associated to a low-immunogenic biocompatiblepolymer, wherein the first leukocyte is allogeneic to the subject; (ii)culturing the first leukocyte with a leukocyte population comprising Tcells from the subject to provide a cultured cellular preparation,wherein the cultured cellular preparation comprises cultured T cellsautologous to the subject; and (iii) administering to the subject atherapeutic amount of the cultured T cells autologous to the subject;wherein the low-immunogenic biocompatible polymer is polyethylene glycol(PEG), or 2-alkyloxazoline (POZ), or hyperbranched polyglycerol (HPG);thereby increasing the ratio of the level of Treg cells to the level ofpro-inflammatory T cells in the subject for treating or alleviating thesymptoms associated with an auto-immune disease afflicting the subject.9. The method of claim 8, wherein the first leukocyte is a T cell. 10.The method of claim 9, wherein the T cell is a CD4-positive T cell or aCD8-positive T cell.
 11. The method of claim 8, further comprisingexpanding the leukocyte population from the subject in vitro prior tostep (ii).
 12. The method of claim 8, wherein the auto-immune disease isat least one of type I diabetes, rheumatoid arthritis, multiplesclerosis, psoriasis, lupus, immune thrombocytopenia, experimentalautoimmune encephalomyelitis, autoimmune uveitis, inflammatory boweldisease, scleroderma and Crohn's disease.
 13. The method of claim 8,wherein the low-immunogenic biocompatible polymer is polyethylene glycol(PEG).
 14. The method of claim 8, wherein the first leukocyte is or theleukocyte population from a subject comprises a peripheral bloodmononuclear cell.
 15. The method of claim 8, wherein the first leukocyteis or the leukocyte population from the subject comprises a splenocyte.16. A method of increasing a ratio of the level of endogenous regulatoryT (Treg) cells to the level of endogenous pro-inflammatory T cells in asubject in need thereof for treating or alleviating the symptomsassociated with an auto-immune disease afflicting the subject, saidmethod comprising: (i) providing a first leukocyte having a cytoplasmicmembrane associated to a low-immunogenic biocompatible polymer, whereinthe first leukocyte is allogeneic to the subject; (ii) culturing thefirst leukocyte with a T cell from the subject to provide a culturedcellular preparation, wherein the cultured cellular preparationcomprises cultured T cells autologous to the subject; and (iii)administering to the subject a therapeutic amount of the cultured Tcells autologous to the subject; wherein the low-immunogenicbiocompatible polymer is polyethylene glycol (PEG), or 2-alkyloxazoline(POZ), or hyperbranched polyglycerol (HPG); thereby increasing the ratioof the level of Treg cells to the level of pro-inflammatory T cells inthe subject for treating or alleviating the symptoms associated with anauto-immune disease afflicting the subject.
 17. The method of claim 16,wherein the first leukocyte is a first T cell.
 18. The method of claim17, wherein the first T cell is a CD4-positive T cell or a CD8-positiveT cell.
 19. The method of claim 16, further comprising expanding the Tcell from the subject in vitro prior to step (ii).
 20. The method ofclaim 16, wherein the auto-immune disease is at least one of type Idiabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, lupus,immune thrombocytopenia, experimental autoimmune encephalomyelitis,autoimmune uveitis, inflammatory bowel disease, scleroderma and Crohn'sdisease.
 21. The method of claim 16, wherein the low-immunogenicbiocompatible polymer is polyethylene glycol (PEG).
 22. The method ofclaim 16, wherein the first leukocyte is a peripheral blood mononuclearcell.
 23. The method of claim 16, wherein the first leukocyte is asplenocyte.